Name: Video: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00
Duration: 14 min 10 s

فإن الكتاب أود أن أشكر ThorLabs لدعمهم لهذا المشروع. AJN تود أن تقر الدعم من زمالة دراسات عليا جبهة الخلاص الوطني. وقد تم تمويل هذا العمل جزئيا من قبل المعاهد الوطنية للصحة من خلال المعاهد الوطنية للصحة المدير الجديد المبتكر برنامج جائزة ومنحة رقم 1 DP2 OD007096 - 01. معلومات عن برنامج مبتكر جديد هو على جائزة <a href="http://nihroadmap.nih.gov/newinnovator/">http://nihroadmap.nih.gov/newinnovator/</a> . فإن الكتاب أود أن أشكر توم هايز لاستخدام مختبر الإلكترونيات في جامعة هارفارد.

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وقد رعت إنتاج هذا الفيديو هذا الفيديو من قبل ، Thorlabs ، وشركة

هذا النظام التصوير بالفيديو معدل يجعل من استخدام نظام تشغيل مرآة galvanometric مدوية في حوالي 8 كيلو هرتز. يمكن أن يكون مرتفعا المرايا مدوية للغاية عندما يعمل في السلطة الكاملة ، والملعب العالية يمكن أن تكون مزعجة أو خطيرة حتى في أوقات التعرض كافية. وإن لم يثبت هنا ، فمن المستحسن لحماية المرآة galvanometric مدوية داخل حقيبة شفافة لخفض كبير في حجم النظام و / أو العتاد المناسب لارتداء السمع وقائية ، مثل سدادات الأذن. المرآة galvanometric بفحص الرنين في نمط الجيبية. ومع ذلك ، وبطاقات framegrabber قراءة في إشارة بافتراض معدل اكتساح خطية تماما في كلا الاتجاهين الأفقي والرأسي. منذ الاجتياح الجيبية يبطئ على حواف الفحص ، يمكن ملاحظة الآثار ضغط الصور على طول محور سريع صورة (أفقي). طريقة واحدة للحد من هذه المشكلة هو محرك عمدا المرآة galvo الرنانة نطاق المسح أكبر بكثير منتتابع عدسة القطر. في القيام بذلك ، لن يؤدي إلا إلى ما يقرب من الاجتياح خطية المركزية لفحص نمط الجيبية تجتاز العينة ، والتقليل من تشوهات الصورة. ونهج آخر يتمثل في ما بعد عملية الصور التي تم جمعها ليصبح خطي المحور بسرعة. ويمكن تحقيق ذلك عن طريق التصوير نمط معروف الفلورية (مثل الشبكة) وباستخدام مقاييس معروفة لخلق نمط نصي unwarps تجهيز الصور التي تم جمعها. وقد صمم هذا النظام مسح خاص لهذا الغرض من التصوير في الجسم الحي ، والتي غالبا ما يتطلب التوجه تستقيم الفيديو معدل المجهر. للتجارب التصوير الخلوية ، وعادة ما تستخدم أكثر المجاهر المقلوب. ويمكن تصميم المقدمة هنا تغييرها بسهولة لبناء مثل هذا المجهر المقلوب ، وكل ما هو مطلوب هو تناوب المباراة النهائية 2 مرآة قطرها &quot;. بدلا من توجيه المرآة لتوجيه شعاع المسح الهبوط ، يمكن للمرآة توجيه شعاع التصاعدي. وضع عدسة هدفر وعلى مسافة واحدة من المرآة جنبا إلى جنب مع مرحلة عينة تسمح للتصوير في الهندسة مقلوب. إذا كان يتم بناء نظام التصوير لمجرد التصوير microendoscopic ، ليس هناك سبب يدعو إلى &quot;اضعاف&quot; تصميم المجهر عموديا على الإطلاق. بدلا من ذلك ، يمكن أن يبنى نظام مسح كامل على اللوح أفقي واحد مع الهدف الموازي عدسة موجهة الى طاولة الضوئية. علما بأن هذا المجهر في بناء التكوين يستخدم الثقب الثابتة ، في حين أن هذا ينص على بناء أكبر بساطة وسهولة التنسيق والمستخدمين الراغبين نظام أكثر تنوعا قد تنظر في إدراج متغير ذات الثقب ، كما يمكن العثور عليها في معظم المجاهر مبائر التجارية. عن طريق السماح للمستخدم لضبط حجم ثقب الدبوس للتعويض عن عينات من كثافة الانبعاثات متفاوتة ، وهذا يسمح للمستخدم لتحسين أفضل لالمفاضلة بين القوة والقرار إشارة لعينة معينة. في الفصلoice من الألياف الصور المختارة للمجهر هو المهم. ونحن نوصي باستخدام الألياف سوميتومو صورة متماسكة نظرا لتباعد الوثيق بينهما الألياف الأساسية وتألق ذاتي نسبي منخفض. تم العثور على صورة ألياف صنعت من قبل فوجيكورا لكميات عالية من تألق ذاتي 10 ، والتي يمكن أن تطغى على إشارات ضعيفة مضان من عينة والحد من حساسية النهائي للmicroendoscope. سوميتومو الألياف المصنعة ، مثل 30N - 8 المستخدمة في هذا الإعداد وجه الخصوص ، لديهم مستويات أقل بكثير من تألق ذاتي معادلاتها فوجيكورا. في حين يمكن اعتبار حزم الألياف يتشيد جذابة للmicroendoscopy ، تصميمها عادة الأماكن النوى الألياف الفردية متباعدة جدا ، وهذا يعني أن الألياف النوى الكائنات عينة قليلة ، تاركة مناطق كبيرة من الفائدة المحتملة. أخيرا ، تجدر الإشارة إلى أنه في حين أن المجهر الموصوفة هنا سيكون مفيدا في مجموعة متنوعة من في المختبر والمجراة للتطبيقيمكن خلق الوظائف ولجزء من تكلفة نظام كامل المواصفات التجارية ، وأنها لا تملك ميزات مثل الكشف عن الضوء المرسل ، والعدسة للعرض ، أو مسار شعاع لepifluorescence widefield غير متحد البؤر. في حين أنه من الممكن بناء نظام مع هذه الميزات من الصفر ، ويمكن للقراء الراغبين مثل هذا النظام ترغب في تعديل نظام قائم التجارية لتلبية احتياجاتهم بدلا من الشروع في بناء جديد تماما.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الجزء </td><td> الصانع </td><td> البند رقم </td><td> مواصفات </td><td> كمية </td></tr><tr><td> 515 نانومتر تصفية باند باس </td><td> صفاء </td><td> HQ515/50M </td><td> 46 FWHM </td><td> 1 </td></tr><tr><td> الوني ضياء دوبليه عدسة 25.4mm. س FL 50mm ، طلاء MgF2 </td><td> ادموند بصريات </td><td> NT49 - 766 </td><td></td><td> 1 </td></tr><tr><td> الوني ضياء دوبليه عدسة 25.4mm. س 76.2mm FL ، طلاء MgF2 </td><td> ادموند بصريات </td><td> NT49 - 768 </td><td></td><td> 1 </td></tr><tr><td> الوني ضياء دوبليه عدسة 25.4mm. س 88.9mm FL ، طلاء MgF2 </td><td> ادموند بصريات </td><td> NT49 - 769 </td><td></td><td> 2 </td></tr><tr><td> الوني ضياء 50mm لنس دوبليه. س 300mm FL ، طلاء MgF2 </td><td> ادموند بصريات </td><td> NT45 - 179 </tد&gt; <td></td><td> 1 </td></tr><tr><td> 8 كيلو هرتز الماسح الضوئي R عالية التردد </td><td> الكهربائية الضوئية منتجات شركة (EOPC) </td><td> SC - 30 </td><td> 8 كيلو هرتز </td><td> 1 </td></tr><tr><td> AGC سائق </td><td> الكهربائية الضوئية منتجات شركة (EOPC) </td><td> ACG : 8K </td><td></td><td></td></tr><tr><td> H7422 - PA Photosensor الوحدة </td><td> هاماماتسو </td><td> H7422 - PA </td><td> أوصت الحد الحالي </td><td> 1 </td></tr><tr><td> M9012 التيار الكهربائي </td><td> هاماماتسو </td><td> M9012 </td><td> للاستخدام مع H7422 - PA </td><td> 1 </td></tr><tr><td> HC PL APO CS الهدف </td><td> لايكا </td><td> 11506284 </td><td> 10x/0.40 </td><td> 1 </td></tr><tr><td> Solios بطاقة Framegrabber EA / XA </td><td> MATROX </td><td> Solios EA / XA </td><td> MIL البرامج المطلوبة ؛ OEM يربط أوصت </td><td> 1 </td></tr><tr><td> 12V التيار الكهربائي </td><tد&gt; Meanwell </td><td> LPV - 100-12 </td><td> +12 V ، 8.5A </td><td> 1 </td></tr><tr><td> 5X عدسة مجهر الهدف </td><td> نيوبورت </td><td> M - 5X </td><td> 0.10 NA ، البعد البؤري 25.4 مم </td><td> 1 </td></tr><tr><td> صورة متماسكة والالياف </td><td> سوميتومو </td><td> 8 - 30N </td><td></td><td> 1 </td></tr><tr><td> 04/01 كاب -20 &quot;برغي وكيت الأجهزة </td><td> ThorLabs </td><td> HW - KIT2 </td><td></td><td> 1 </td></tr><tr><td> الخيالة 100 ميكرون الثقب </td><td> ThorLabs </td><td> P100S </td><td> مثالية لبناء مصافي المكانية </td><td> 1 </td></tr><tr><td> 30 ملم مكعب كيج المشبك </td><td> ThorLabs </td><td> B6C </td><td></td><td> 1 </td></tr><tr><td> 30 ملم مكعب نظام كيج ، 4 - الطريق </td><td> ThorLabs </td><td> C4W </td><td></td><td> 1 </td></tr><tr><td> 406 نانومتر ، 5 ميغاواط ، B رمز PIN ، SM ديود الليزر والالياف Pigtailed ، FC / PC </td><td> ThorLabs </td><td> LPS - 406 - FC </td><td> عفا عليها الزمن المنتج ؛ استبداله SF10 - LP405 </td><td> 1 </td></tr><tr><td> 5 - اللحظة الايبوكسي ، 1 اوقية (الاونصة) </td><td> ThorLabs </td><td> G14250 </td><td></td><td> 1 </td></tr><tr><td> 6 محور الحركية البصرية جبل </td><td> ThorLabs </td><td> K6X </td><td></td><td> 1 </td></tr><tr><td> 8-32 برغي كاب ومجموعة الأجهزة </td><td> ThorLabs </td><td> HW - KIT1 </td><td></td><td> 1 </td></tr><tr><td> 8-32 الصامولة وكيت الأجهزة </td><td> ThorLabs </td><td> HW - KIT3 </td><td></td><td> 1 </td></tr><tr><td> محول مع RMS المواضيع الداخلية والخارجية المواضيع SM1 </td><td> ThorLabs </td><td> SM1A4 </td><td></td><td> 1 </td></tr><tr><td> صفة. FC / PC وتلسكوب الموازاة FC / APC ، و = 2.0 ملم ، مركز البحوث الزراعية : 400-600 نانومتر </td><td> ThorLabs </td><td> CFC - 2X - A </td><td> و = 2.0 مم </td><td> 1 </td></tr><tr><td> تعديل محول الألياف تلسكوب الموازاة ، SM1 خيوط </td><td> ThorLabs </td><td> AD9.5F </td><td></td><td> 1 </td></tr><tr><td> الألمنيوم اللوح ، 12 &quot;× 18&quot; س 1 / 2 &quot; </td><td> ThorLabs </td><td> MB1218 </td><td> 1 / 4 &quot;-20 خيوط </td><td> 2 </td></tr><tr><td> الفوق ليزر ديود / المراقب TEC </td><td> ThorLabs </td><td> ITC4001 </td><td> 1 A/96 W </td><td> 1 </td></tr><tr><td> DMLP 425 نانومتر ممر طويل ميرور مزدوج اللون </td><td> ThorLabs </td><td> DMLP425 </td><td></td><td> 1 </td></tr><tr><td> جبل الحركية للبصريات &quot;O1 </td><td> ThorLabs </td><td> KM100 </td><td></td><td> 3 </td></tr><tr><td> LD / TEC جبل ليزر الثنائيات ThorLabs الألياف Pigtailed </td><td> ThorLabs </td><td> LM9LP </td><td></td><td> 1 </td></tr><tr><td> عدسة جبل لØ18 ملم بصريات </td><td> ThorLabs </td><td> LMR18 </td><td> وشملت حلقة واحدة الاحتفاظ </td><td> 1 </td></tr><tr><td> عدسة المحرك للبصريات &quot;(2) </td><td> ThorLabs </td><td> LMR2S </td><td> مع خيوط الداخلية والخارجية ، واشتملت على عصابة التجنيب </td><td> 2 </td></tr><tr><td> مصغرة سلسلة قفص الجمعية رود ، 6 &quot;لونغ ، O4 ملم ، والكمية. 1 </td><td> ThorLabs </td><td> SR6 </td><td></td><td> 4 </td></tr><tr><td> Ø1.0 &quot;آخر عمود الركيزة ، 8-32 الحنفيات ، 1&quot; لونج </td><td> ThorLabs </td><td> RS1P8E </td><td></td><td> 4 </td></tr><tr><td> O1 آخر عمود &quot;ملحق ، المدة = 0.5 </td><td> ThorLabs </td><td> RS05 </td><td></td><td> 4 </td></tr><tr><td> O1 &quot;آخر عمود ملحق ، المدة = 0.75&quot; </td><td> ThorLabs </td><td> RS075 </td><td></td><td> 4 </td></tr><tr><td> O1 &quot;محمية سيلفر ميرور ، 3.2 مم </td><td> ThorLabs </td><td> ME1 - P01 </td><td></td><td> 1 </td></tr><tr><td> O1 &quot;SM1 الدوارة تعديل عنصر التركيز ، L = 1&quot; </td><td> ThorLabs </td><td> SM1V10 </tد&gt; <td></td><td> 1 </td></tr><tr><td> O2 &quot;محمية سيلفر ميرور ، 3.2 مم </td><td> ThorLabs </td><td> ME2 - P01 </td><td></td><td> 2 </td></tr><tr><td> الخيالة Ø100 ميكرومتر الثقب -- P100S </td><td> ThorLabs </td><td> P100S </td><td></td><td> 1 </td></tr><tr><td> بولاريس الانجراف قليلة O1 &quot;ميرور جبل الحركية </td><td> ThorLabs </td><td> POLARIS - K1 </td><td> انخفاض العائمة </td><td> 1 </td></tr><tr><td> SM1 انس الأنبوب ، L = 1 &quot; </td><td> ThorLabs </td><td> SM1L - 10 </td><td> وشملت حلقة واحدة الاحتفاظ </td><td> 4 </td></tr><tr><td> SM1 كيج 30 ملم خيوط اللوحة ، 0.35 &quot;سميكة </td><td> ThorLabs </td><td> CP02 </td><td></td><td> 2 </td></tr><tr><td> SM1 إلى M25 محول المكونات البصرية التخييط </td><td> ThorLabs </td><td> SM1A24 </td><td> المواضيع الخارجية والداخلية SM1 المواضيع M25.5x0.5 </td><td> 1 </td></tr><tr><td> الحزمة الصغيرة ديامالعطر Galvo النظام </td><td> ThorLabs </td><td> GVSM001 </td><td></td><td> 1 </td></tr><tr><td> شوكة صغيرة المشبك </td><td> ThorLabs </td><td> CF125 </td><td> 1 / 25 &quot;counterbored فتحة عالمية </td><td> 15 </td></tr><tr><td> تصفية نظام المكانية </td><td> ThorLabs </td><td> KT310 </td><td> تباع بشكل منفصل الثقب </td><td> 1 </td></tr><tr><td> TE - تبريد جبل ليزر 5.6 و 9 ملم </td><td> ThorLabs </td><td> TCLDM9 </td><td></td><td> 1 </td></tr><tr><td> القوس الرأسية للالألواح </td><td> ThorLabs </td><td> VB01 </td><td> كل </td><td> 2 </td></tr><tr><td> خطة لامزيغ - </td><td> زايس </td><td> 1101-957 </td><td> 20x/0.75 NA </td><td> 1 </td></tr></tbody></table>

video-rate scanning confocal microscopy and microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

يوصف البناء كاملة مخصصة ، في الوقت الحقيقي المسح مبائر نظام التصوير. هذا النظام ، والتي يمكن استخدامها بسهولة لمعدل الفيديو وmicroendoscopy المجهري ، ويسمح لمجموعة من التطبيقات وهندستها التصوير لا يمكن الوصول إليها باستخدام معيار نظم مبائر التجارية ، في جزء صغير من التكلفة.

فيديو معدل الميكروسكوب متحد البؤر المسح الضوئي وMicroendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

video-rate-scanning-confocal-microscopy-and-microendoscopy

Research

JoVE Journal

Bioengineering

27.6K Views.  Harvard University. يوصف البناء كاملة مخصصة ، في الوقت الحقيقي المسح مبائر نظام التصوير. هذا النظام ، والتي يمكن استخدامها بسهولة لمعدل الفيديو وmicroendoscopy المجهري ، ويسمح لمجموعة من التطبيقات وهندستها التصوير لا يمكن الوصول إليها باستخدام معيار نظم مبائر التجارية ، في جزء صغير من ا?...

Video: Video-rate Scanning Confocal Microscopy and Microendoscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

ذات دقة عالية الألياف الضوئية للMicroendoscopy في الموقع الخلوية التصوير

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

منخفضة التكلفة المرحلة الميكروسكوب البرد الخفيفة التصنيع الخفيف مربوط / مجهرية إلكترون

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

تحضير العينة ، والتصوير ، وتحليل بروتوكولات لالميكروسكوب الضوئي سكين الحافة

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

المتلازم الخفيفة والمجهر الإلكتروني عن طريق الكم دوت النانوية

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

المتعدد الوسائط التصوير ومنهاج Microendoscopy الطيفي الألياف الرزمة لغير الغازية،<em&gt; في فيفو</em&gt; تحليل الأنسجة

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

تشريح، المسح الضوئي MicroCT والمظهرية تحليلات من جذيل

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

انقسام الخلية الثديية في مصفوفات ثلاثية الأبعاد عن طريق الفحص المجهري انعكاس [كنفوكل] الكمية

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

النقل المتعدد الوسائط "الشبكية الحجمي التصوير المائل المسح الضوئي الليزر تنظير العين" (أوسلو) و "التصوير المقطعي التماسك الضوئية" (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

تصور تشكيل الالتصاق في الخلايا من خلال متقدم الغزل مجهرية Fluorescence انعكاس الداخلية القرص--المجموع

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

قرب متزامنة ليزر المسح Confocal والقوة الذرية المجهرية (Conpokal) على الخلايا الحية

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...