Imaging Biological Samples with Optical and Confocal Microscopy

Panoramica

Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut

Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal conditions. However, optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. While the resolution of optical microscopes does not reach that of electron microscopes, they are the most valuable tools in the imaging of biological macrostructures and are a staple in any biological lab.

In conventional light microscopes, the signal produced from the imaged object is from the full thickness of the specimen, which does not allow most of it to be in focus to the observer. This causes the image to have "out of focus blur". The confocal microscope, on the other hand, illuminates the sample through a pin-hole, and is therefore able to filter out the out-of-focus light from above and below the point of focus in the object.

This demonstration provides an introduction to image acquisition using optical and confocal microscopy methods. Here, a sectioned piece of mouse brain will be studied. Image acquisition and analysis, including the tools to generate topographical maps and composite images, will be covered. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to provide more information on optical and confocal microscopes to determine if these microscopy modules are the best fit for a type of biological sample.

Procedura

1. Confocal Imaging

Load the sample onto the stage. Center it beneath the lens. It should not exceed the weight limitation of the stage, which in this case is 5 kg. The sample should not be more than 100 mm thick.
Open the imaging software and select "Create Job."
Under the Topographies Column, choose the assistant button.
Create an overview image at the lowest magnification, 2.5X. Before switching the magnifications, ensure that the sample is in focus by changin

Risultati

The following images give an overview of results that can be obtained of a mouse brain using a confocal microscope. They show how varying levels of information can be obtained and how a topographical map of the results reveals the height of the sample.

Figure 5: Confocal images at

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Applicazione e Riepilogo

In this demonstration, the depth of focus, field of view and maximum resolution and magnification of optical and confocal microscopes were optimized to view biological samples. This demonstration was designed to help the participant decide which microscopy module is the best for a certain application. Both modes of microscopy have advantages in analyzing biological samples for their ease of preparation and high resolution composite images.

The applications for optical and confocal microscopy a

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1. Confocal Imaging

Load the sample onto the stage. Center it beneath the lens. It should not exceed the weight limitation of the stage, which in this case is 5 kg. The sample should not be more than 100 mm thick.

Open the imaging software and select "Create Job."

Under the Topographies Column, choose the assistant button.

Create an overview image at the lowest magnification, 2.5X. Before switching the magnifications, ensure that the sample is in focus by changing the Z position until you see a clear image. This can be done by pushing down or pulling up on the 3D microscope manipulator. Finer Z motion is attained by engaging the button on the side, which causes a blue light around the edges.

Slowly increase the lens magnification, continuously playing with the light intensity and the focus until you are at the desired magnification. If desired, choose a different area of interest by moving the stage in the x and y directions by using the manipulator.

Once an overview image is taken at the low magnification, hit the next button to proceed to the Reference Point step. If desired, specify a certain reference point for the sake of measurement (i.e., the corner of the sample), though for this purpose the default reference point is fine.

Hit the next arrow to proceed to the next part of the wizard.

Change the objective as desired to see a resolution appropriate for your sample. In this case, the 50X objective lens is used to visualize the cells in the sample. The 50X is the lens that is closest to the sample, so gradually move up to the 50X objective ensuring that there is still room to decrease the working distance after the 20X lens.

On the "Measuring Range Definition" page, move the Z position slightly (clicking the side button on the manipulator for fine adjustments) so that only the very top of the sample is in focus and click "Set Last". Then move the stage in the Z direction (downwards) until only the very bottom of the sample is in focus and press "Set First". Make sure that the number of calculated slices does not exceed 1000 or the program will fail.

Make sure that the intensity of light does not oversaturate (cause red pixels) in the image at any of the levels and then hit done. This will take the tomography image and open the tomography software.

In the tomography software, open tabs such as the Studies tab to view the data in 3D and take measurements in 2D or 3D space.

2. Digital Optical Microscope Imaging

Load the sample onto the stage. Center the sample beneath the lens. The sample weight should not exceed the weight limitation of the stage, which in this case is 4 kg. The sample should be no higher than 12 cm.

Open the imaging software.

Select a Job from the list of templates provided. It is also possible to work outside of a job by pressing Free Examination, which allows you to study a sample outside of a job.

Acquire an overview image that shows the entire stage. This will serve as a map later on to display the part of the sample that is being viewed. While acquiring the image, use the controller to change the focus and the image position.

Place a coordinate system. The default coordinate system is from the back-left corner of the stage and is fine for this application. If the sample is crooked, you can adjust the coordinates here.

Name the sample and the job, this adds it to the list of jobs so that other users can return to it.

Select the camera button under Acquire. Take an initial image, and then press the live button as you navigate the sample.

Move the focus downwards until the sample is clearly in focus. It may be necessary to also adjust the lighting under the Lighting and Aperture" tab.

Optimize the image with the tools under the Image Optimization panel. You can play with different parameters under the Image Enhancements tab, such as the tilt of the lens, the lighting levels on the sample, and brightness and contrast, until the image has the desired crispness.

Perform measurements by tapping the Pencil tool on the software. From there, you have access to several measurement tools, including distances, angles, and area. Use the distance and area tools to measure the size of the sample.

Go to the Results Workflow tab and check the workflow layout to configure the layout of the report

Tap the save button to save your job so that others can use the same workflow.

Vai a...

0:07

Overview

1:26

Principles of Confocal Microscopy

3:37

Confocal Imaging

6:09

Digital Optical Microscope

7:56

Results

9:05

Applications

10:16

Summary

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