We would like to thank D. Stephens (University of Bristol, Bristol, United Kingdom) for the TPST1-EGFP DNA plasmid, as well as Lakshmi Narasimhan Govindarajan for helping with the software optimization. This work was supported by grants from the National Medical Research Council (NMRC/CBRG/007/2012), Ministry of Education (AcRF Tier1 RG 18/11, RG 48/13 and RG132/15 and AcRF Tier2 MOE2015-T2-2-073) to L.L.

Note: Below is a step-by-step protocol of GLIM for determining the LQ of EGFP-tagged tyrosylprotein sulfotransferase 1 (TPST1), a Golgi resident enzyme, in HeLa cells.
1. Preparation of Fluorescence-labeled Golgi Mini-stacks
<ol>
	<li>Prepare glass coverslips
	<ol>
		<li>Aliquot 0.3 mL sterile Dulbecco Modified Eagle&#39;s Medium (DMEM) to a well of a 24-well plate in a tissue culture hood.</li>
		<li>Wipe a piece of &#934; 12 mm No.1.5 glass coverslip using soft tissue pape...

<ol>
	<li>Glick, B. S., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+Golgi+traffic:+a+critical+assessment.">Models for Golgi traffic: a critical assessment.</a> Cold Spring Harb Perspect Biol. 3 (11), 005215 (2011).</li><li>Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+mammalian+Golgi.">Architecture of the mammalian Golgi.</a> Cold Spring Harb Perspect Biol. 3 (7), (2011).</li><li>Lu, L., Hong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+endosomes+to+the+trans-Golgi+network.">From endosomes to the trans-Golgi network.</a> Semin Cell Dev Biol. , (2014).</li><li>De Matteis, M. A., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exiting+the+Golgi+complex.">Exiting the Golgi complex.</a> Nat Rev Mol Cell Biol. 9 (4), 273-284 (2008).</li><li>Lu, L., Ladinsky, M. S., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisternal+organization+of+the+endoplasmic+reticulum+during+mitosis.">Cisternal organization of the endoplasmic reticulum during mitosis.</a> Mol Biol Cell. 20 (15), 3471-3480 (2009).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> J Cell Biol. 190 (2), 165-175 (2010).</li><li>Tie, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+imaging+method+for+quantitative+Golgi+localization+reveals+differential+intra-Golgi+trafficking+of+secretory+cargoes.">A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes.</a> Mol Biol Cell. 27 (5), 848-861 (2016).</li><li>Trucco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+traffic+triggers+the+formation+of+tubular+continuities+across+Golgi+sub-compartments.">Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments.</a> Nat Cell Biol. 6 (11), 1071-1081 (2004).</li><li>Cole, N. B., Sciaky, N., Marotta, A., Song, J., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+dispersal+during+microtubule+disruption:+regeneration+of+Golgi+stacks+at+peripheral+endoplasmic+reticulum+exit+sites.">Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.</a> Mol Biol Cell. 7 (4), 631-650 (1996).</li><li>Rogalski, A. A., Bergmann, J. E., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+microtubule+assembly+status+on+the+intracellular+processing+and+surface+expression+of+an+integral+protein+of+the+plasma+membrane.">Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.</a> J Cell Biol. 99 (3), 1101-1109 (1984).</li><li>Van De Moortele, S., Picart, R., Tixier-Vidal, A., Tougard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocodazole+and+taxol+affect+subcellular+compartments+but+not+secretory+activity+of+GH3B6+prolactin+cells.">Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.</a> Eur J Cell Biol. 60 (2), 217-227 (1993).</li><li>Nakamura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+cis-Golgi+matrix+protein,+GM130.">Characterization of a cis-Golgi matrix protein, GM130.</a> J Cell Biol. 131, 1715-1726 (1995).</li><li>Roth, J., Berger, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+localization+of+galactosyltransferase+in+HeLa+cells:+codistribution+with+thiamine+pyrophosphatase+in+trans-Golgi+cisternae.">Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.</a> J Cell Biol. 93 (1), 223-229 (1982).</li><li>Baeuerle, P. A., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+sulfation+is+a+trans-Golgi-specific+protein+modification.">Tyrosine sulfation is a trans-Golgi-specific protein modification.</a> J Cell Biol. 105 (6), 2655-2664 (1987).</li><li>Honda, A., Al-Awar, O. S., Hay, J. C., Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Arf-1+to+the+early+Golgi+by+membrin,+an+ER-Golgi+SNARE.">Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE.</a> J Cell Biol. 168 (7), 1039-1051 (2005).</li><li>Lavieu, G., Zheng, H., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stapled+Golgi+cisternae+remain+in+place+as+cargo+passes+through+the+stack.">Stapled Golgi cisternae remain in place as cargo passes through the stack.</a> Elife. 2, 00558 (2013).</li><li>Zhao, X., Lasell, T. K., Melancon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+large+ADP-ribosylation+factor-guanine+nucleotide+exchange+factors+to+different+Golgi+compartments:+evidence+for+distinct+functions+in+protein+traffic.">Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.</a> Mol Biol Cell. 13 (1), 119-133 (2002).</li><li>Dejgaard, S. Y., Murshid, A., Dee, K. M., Presley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy-based+linescan+methodologies+for+intra-Golgi+localization+of+proteins.">Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins.</a> J Histochem Cytochem. 55 (7), 709-719 (2007).</li><li>Fourriere, L., Divoux, S., Roceri, M., Perez, F., Boncompain, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-independent+secretion+requires+functional+maturation+of+Golgi+elements.">Microtubule-independent secretion requires functional maturation of Golgi elements.</a> J Cell Sci. 129 (17), 3238-3250 (2016).</li><li>Volchuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Countercurrent+distribution+of+two+distinct+SNARE+complexes+mediating+transport+within+the+Golgi+stack.">Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.</a> Mol Biol Cell. 15 (4), 1506-1518 (2004).</li><li>Popoff, V., Adolf, F., Brugger, B., Wieland, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+budding+within+the+Golgi+stack.">COPI budding within the Golgi stack.</a> Cold Spring Harb Perspect Biol. 3 (11), 005231 (2011).</li></ol>

Previously, the localization of a Golgi protein under the light microscopy was mainly quantified by the degree of correlation or overlapping of the image of the protein with the image of a Golgi marker of known localization15,16,17. The resulting correlation or overlapping coefficient reflects how close the testing protein is to the Golgi marker spatially. There are at least three caveats for this approach. First, the correlatio...

The Golgi complex plays essential roles in secretory/endocytic trafficking of proteins and lipids (hereafter cargos) in mammalian cells1,2,3. At the Golgi, cargos are not only sorted to various sub-cellular compartments but also modified by diverse types of glycosylation. The mammalian Golgi complex comprises numerous laterally connected Golgi stacks, which typically consists of 4 - 11 tightly adjacent and flat membrane sacs called cisternae. The serially stacked Golgi cisternae are further categorized, from one end to the other, as cis, medial and trans-cisternae. At the trans-side of a Golgi stack, the trans-most membrane sac develops into a tubular and reticulum membrane network called the trans-Golgi network (TGN)4. In the secretory pathway, cargos derived from the endoplasmic reticulum (ER) enter a Golgi stack at its cis-side and then sequentially pass through medial and trans-cisternae. Cargos eventually exit the Golgi at the trans-Golgi or TGN destining to the plasma membrane, endosomes or secretory granules.
The molecular and cellular mechanisms of how cargos transit a Golgi stack and how the Golgi maintains its cisternal organization remain mysterious and are currently still under a heated debate1. One of difficulties in this field is that Golgi cisternae can only be resolved under the electron microscopy (EM) since the resolution of an optical microscope (~ 200 nm) is insufficient to resolve individual Golgi cisternae (&#60; 100 nm in both cisternal thickness and distance). Therefore, the sub-Golgi localization of resident proteins and transiting cargos are conventionally determined by the immuno-gold EM. However, the immuno-gold EM is very technically demanding and it is beyond the capability of most cell biology labs. Although the resolution of the EM can be sub-nanometer, the resolution afforded by the immuno-gold EM is greatly hampered by the size of the antibody complex (primary plus the secondary antibody) and the gold particle, and it can be worse than 20 nm. Furthermore, EM images are obtained from 2D thin-sections instead of a 3D global view of the Golgi, which can result in erroneous conclusions depending on the relative position and orientation of the 2D section5. For example, studying an EM single-section is unable to reliably differentiate a vesicle from the orthogonal view of a tubule since both can display identical round membrane profiles. The recent advent of super-resolution microscopy techniques, such as 3D-structured illumination microscopy (3D-SIM), stimulated emission depletion (STED), photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), makes it possible to resolve sub-Golgi structures under light microscopes6. However, there are at least four drawbacks that can significantly limit their uses in the cell biological study of the Golgi. 1) Current super-resolution techniques require expensive and special hardware configuration which is beyond most cell biology labs. 2) Special fluorescence labeling protocols are needed for some super-resolution techniques. 3) Although, under the best condition, these techniques claim 20-110 nm in spatial resolution, the practical resolution obtained in real samples can be much worse. 4) In comparison to conventional microscopy, these super-resolution techniques still have difficulties in conducting multicolor, 3D or live cell imaging, either singly or in combination. Probably most importantly, both immuno-gold EM and the super-resolution microscopy techniques yield qualitative instead of quantitative localization data.
Attempting to partially solve problems mentioned above, we have recently developed a conventional light microscopy based method, which is named Golgi protein localization by imaging centers of mass (GLIM), to systematically and quantitatively localize a Golgi protein at a resolution equivalent to that of the immuno-gold EM7. In this method, the Golgi in cultured mammalian cells is dispersed as Golgi mini-stacks by the treatment of nocodazole, a microtubule depolymerizing drug. Extensive studies have demonstrated that nocodazole-induced Golgi mini-stacks (hereafter Golgi mini-stacks) closely resemble native Golgi stacks in both organization and cellular functions8,9,10,11. The localization quotient (LQ) of a test protein can be acquired through GLIM and it denotes the quantitative sub-Golgi localization. The numerical values of LQs can be compared and a LQ database of more than 25 Golgi markers has been available.
In GLIM, Golgi mini-stacks are triple-labeled by endogenous or exogenously expressed GM130, GalT-mCherry and the test protein (x). GM130 and GalT-mCherry, cis- and trans-Golgi markers respectively12,13, provide reference points. The triple fluorescence, red (R), green (G) and far-red (B), are artificially displayed as red, green and blue, respectively. Center of fluorescence mass (hereafter center) is adopted to achieve sub-pixel resolution. The Golgi axis is defined as the vector from the center of GM130 to that of GalT-mCherry. The Golgi mini-stack is modeled as a cylindrical structure with infinite rotational symmetry around the Golgi axis. Therefore, a Golgi mini-stack can be further modeled as an one-dimensional structure along the Golgi axis. The LQ of the test protein x is defined as dx/d1, in which dx is the distance from the center of x to that of GM130, while d1 is the distance from the center of GalT-mCherry to that of GM130. If the center of x is off-axis, its projection axial distance is used for the calculation. The variables, including Golgi axis, axial angle, dx, d1, angle &#945; and angle &#946;, for GLIM are schematically illustrated in Figure 1. LQ is independent of the Golgi axial angle though Golgi mini-stacks orient randomly in a cell.
Golgi mini-stacks appear inhomogeneous in images. We developed three criteria to select analyzable Golgi mini-stacks for GLIM. 1) The signal-to-noise ratio criterion, in which the ratio of the total intensity of a Golgi mini-stack to the standard deviation (SD) of the background is &#8805; 30 in each channel. This criterion is to ensure the positioning accuracy of the center of mass, which depends on the signal-to-noise ratios of Golgi mini-stacks. 2) The axial angle or distance criterion, which requires d1&#8805; 70 nm. d1 decreases with the increase of the Golgi axial angle. When the axial angle is approaching 90&#176; or vertical, the mini-stack becomes non-resolvable as d1 is approaching 0. d1&#8805; 70 nm can effectively exclude near vertical Golgi mini-stacks. 3) The co-linearity criterion, in which either |tan &#945;| or |tan &#946;| is &#8804; 0.3. This criterion ensures that the three centers of a mini-stack are sufficiently co-linear for our one-dimensional model of the Golgi mini-stack. All light microscopes suffer from chromatic aberration which can seriously distort the relative positions of red, green and far-red fluorescence centers. Chromatic aberration of microscope systems is experimentally calibrated by imaging 110 nm beads, which are triple-labeled by red, green and far-red fluorescence. For each bead image, the center of red is defined as the true position of the bead and chromatic-shifts of green and far-red centers are fitted by first-order polynomial functions. Centers of Golgi mini-stacks are subjected to the polynomial functions to correct the chromatic-shifts in green and far-red channels.
Through GLIM we can achieve a resolution of ~ 30 nm along the Golgi axis under standard conditions. Importantly, it provides a systematical method to quantitatively map any Golgi protein. GLIM can be performed by conventional microscopes, such as wide-field or confocal microscopes, using common fluorescence labeling protocols. The imaging and data processing can take as short as an hour. Through GLIM, we have directly demonstrated the progressive transition of the secretory cargo from the cis- to trans-side of the Golgi7.

<table>
	<tbody>
		<tr>
			<td class="xl67">fluorescence beads. Commercial name: TetraSpeck beads</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">T7279</td>
			<td class="xl67">As multi-color beads to calibrate chromatic-shift of the microscope.</td>
		</tr>
		<tr>
			<td class="xl67">Glass coverslip &#934; 12 mm (No. 1.5)</td>
			<td class="xl67">Menzel</td>
			<td class="xl75">CB00120RAC</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Glass coverslip &#934; 25 mm (No. 1.5)</td>
			<td class="xl67">Menzel</td>
			<td class="xl75"></td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">DMEM</td>
			<td class="xl67">Capricon</td>
			<td class="xl75">DMEM-HPA-P50</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Trypsin-EDTA</td>
			<td class="xl67"></td>
			<td class="xl75"></td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">FBS</td>
			<td class="xl67">GE Hyclone</td>
			<td class="xl75">SV30160.03</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Nocodazole</td>
			<td class="xl67">Merck</td>
			<td class="xl75">487928</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">transfection reagent. Commercial name: Lipofectamine 2000</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">11668-019</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">transfection medium. Commercial name: OptiMEM</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">31985070</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">TPST1-EGFP</td>
			<td class="xl67">Addgene</td>
			<td class="xl75">66617</td>
			<td class="xl67">A gift from D. Stephens (University of Bristol, Bristol, United Kingdom)</td>
		</tr>
		<tr>
			<td class="xl67">GalT-mCherry</td>
			<td class="xl67"></td>
			<td class="xl75"></td>
			<td class="xl67">Made in our lab.</td>
		</tr>
		<tr>
			<td class="xl67">paraformaldehyde</td>
			<td class="xl67">Merck</td>
			<td class="xl75">1.04005.1000</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">saponin</td>
			<td class="xl67">Sigma-Aldrich</td>
			<td class="xl75">47036</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">poly(vinyl alcohol) (Mw ~31,000). Commercial name: Mowiol-488</td>
			<td class="xl67">CALBIOCHEM</td>
			<td class="xl75">475904</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">BSA</td>
			<td class="xl67">Sigma-Aldrich</td>
			<td class="xl75">A9647</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Mouse anti-GM130</td>
			<td class="xl67">BD Biosciences</td>
			<td class="xl75">610823</td>
			<td class="xl67">Primary antibody for human GM130</td>
		</tr>
		<tr>
			<td class="xl67">far-red fluorescence conjugated goat anti-mouse IgG. Commercial name: Alexa Fluor 647 conjugated goat anti-mouse IgG</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">A-21235</td>
			<td class="xl67">Far red fluorescence conjugated secondary antibody</td>
		</tr>
	</tbody>
</table>

quantitative localization of a golgi protein by imaging its center of fluorescence mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The modern research grade light microscope equipped with a plan apochromatic lens, such as the one used in our lab, shows minimal chromatic aberration (Figure 2A). However, a careful examination of the multi-color fluorescent bead image can reveal the shift of different color images of the same bead (Figure 2B). We define that the red channel is free of chromatic aberration and therefore centers ...

Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

The overall goal of this imaging-based method is to quantitatively localize a golgi protein at nanometer resolution. This method can help to answer the key questions in the study of the golgi apparatus such as the golgi's organization and how it works. The main advantage of this method is that it uses a conventional light microscope and software tools.To begin, culture HeLa cells in a T25 flask in DMEM supplemented with 10%FBS and incubate the culture at 37 degrees Celsius and 5%carbon dioxide. When cells reach approximately 80%confluency, aspirate the culture medium and add one milliliter of 25%Trypsin EDTA to the flask. Incubate the cells at 37 degrees Celsius for two minutes.Remove the cells from the incubator and add one milliliter of complete medium to gently flush the cells off the wall of the flask. Transfer the detached cells to a sterile centrifugation tube and spin the tube at 500 g's for two minutes to pellet the cells. After aspirating the supernatant, use one milliliter of complete medium to suspend the cell pellet.Remove the medium from the well of a 24-well plate containing a sterilized glass cover slip and seed approximately 100, 000 cells into the well. Use complete medium to top up the volume of the well to 5 milliliters. Then, incubate the plate at 37 degrees Celsius with 5%carbon dioxide.When the cells have reached 80%confluency, use 80 nanograms of GalT-mCherry and 320 nanograms of TPST1-EGFP plasmid DNA and transfection reagent to transfect the cells according to the manufacturer's protocol. After incubating the cells for four to six hours, change the medium and return the cells to the incubator for 12 hours. Aspirate the medium in the well and add 5 milliliters of 33 micromold Nocodazole containing complete medium and then incubate the cells at 37 degrees Celsius and 5%carbon dioxide for three hours.After cleaning glass cover slips according to the text protocol, use one times PBS containing 1 microgram per microliter of BSA to dilute 110 nanometers multi-color fluorescent beads 80 fold. Briefly vortex the tube to disperse the bead aggregates. Using a pipette tip, spread 60 microliters of diluted beads onto a clean cover slip.In the dark, place the cover slip in a 35 millimeter dish and cover it with aluminum foil. Dry the cover slip in the dark in a desiccator connected to a vacuum pump. Then use 50 microliters of mounting medium to mount the cover slip onto a glass slide.Properly clean cover slips are essential to eliminate fluorescent debris. To image multi-color beads in green, red, and far red channels, at the beginning of the imaging session, acquire 3D stacks of images, taking three sections above and below the best focal plane. Save the stacks as TIFF files.To image golgi mini-stacks, use TPST1-EGFP and GalT-mCherry transfected in fluorescently labeled slides to find cells that express TPST1-EGFP and a low or medium level GalT-mCherry. Acquire 3D image stacks as just demonstrated. To analyze the images, open image J, choose File, Image, Open, to open a set of bead images consisting of three TIFF files.Choose Image, Stacks, Z Project, to average three consecutive sections around the best focused section in the red channel. Input the section number of start slice and stop slice and among options of projection type, select average intensity. Draw background ROIs that contain no beads in the image.Then in Analyze, Set Measurements, check only mean gray value and Standard Deviation or SD.Execute Analyze, Measure, to obtain the background mean intensity and standard deviation. Choose Process, Math, Subtract, to subtract the image with the corresponding values of the background mean intensity plus six times the standard deviation. Then input the calculated value corresponding to this channel.Repeat the averaging and subtraction for green and far red channel image stacks by using the same start and stop slice and the same background ROI. Merge the three processed images using Image, Color, Merge Channels, by selecting channel R as red, channel G as green, and channel B as blue. Launch the ROI Manager by choosing Analyze, Tools, ROI Manager.Draw square ROIs around single beads. Then add the ROI to the ROI Manager by pressing T on the keyboard. Under Analyze, Set Measurements, check only center of mass.In the ROI Manager, select channel R and click Measure to obtain the X and Y coordinates of the centers of ROIs in the results window. Copy and paste the two columns into a spreadsheet. Obtain the coordinates of the centers of ROIs for channel G and channel B.Save the spreadsheet as beads.csv.In image J, use plugins install to install the two macros, macro golgi ROI inspection and macro output three channels data. Clear the ROI Manager and results window. Open a set of golgi mini-stack images consisting of three TIFF files.Generate background subtracted images as demonstrated earlier in this video for later use. Then duplicate the images. Under Process, Image Calculator, add the background subtracted channel G, channel R, and channel B images.To the resulting image under Image, Adjust, Threshold, select Set to input one as the lowest threshold level. Under Analyze, Analyze Particles, input the size range for size pixel too. Input 50 to infinity, check excludes on edges, and add to manager.Next, use Image, Color, Merge Channels, to merge the three background subtracted images generated earlier by selecting channel R as red, channel G as green, and channel B as blue. Then run the macro macro golgi ROI inspection by selecting Plugins, Macro, Golgi ROI Inspection. It is important to select as many ROIs as possible that contains single golgi mini-stacks in a set of images.Under Analyze, Set Measurements, check only area, mean gray value, and center of mass. Acquire data by launching the macro tool macro output three channels data. Copy coordinates of centers into a spreadsheet in the order listed here.Then save the spreadsheet as ministacks.csv. Proceed to chromatic shift correction of the centers and Localization Quotient or LQ calculation according to the text protocol. The modern research grade light microscope equipped with the plain apochromatic lens such as the one used here shows minimal chromatic shift.However, a careful examination of the multi-colored bead image reveals it. Centers of red fluorescence are defined as the true positions of beads. The relative chromatic shifts of green and far red fluorescence can be represented by vectors.The chromatic shift within the imaging plane is illustrated by the green and blue vector for each bead. As illustrated here, chromatic shift correction significantly reduces the chromatic shift. In mammalian cells, the golgi complex is aggregated at the perinuclear area which is usually not resolvable under a conventional optical microscope.After Nocodazole treatment, the perinuclear golgi complex disappears and dozens of golgi mini-stacks assemble at the endoplasmic reticulum exit sites throughout the cytoplasm. An example image that was generated using the image analysis demonstrated in this video is shown here. Using the macro golgi ROI inspection tool, 40 selected golgi mini-stacks were listed.After applying the three criteria, 21 golgi mini-stacks were analyzable for the calculation of LQs of TPST1-EGFP. Once mastered, this technique can be done in one hour after fluorescence labeling. After its development, this method paved the way for researchers who study the golgi apparatus to quantitatively localize a golgi protein with resolution equivalent to that of immunoelectron microscopy.

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Research

JoVE Journal

Biology

10.6K visualizações.  Nanyang Technological University. The overall goal of this imaging-based method is to quantitatively localize a golgi protein at nanometer resolution. This method can help to answer the key questions in the study of the golgi apparatus such as the golgi's organization and how it works. The main advantage of this method is that it uses a conventional light microscope and software tools.To begin, culture HeLa cells in a T25 flask in DMEM supplemented with 10%FBS and incubate the culture at 37 degrees Celsius and 5%carbon...