1St Annual Undergraduate Quantitative Biology Summer School Module 1: Single-Cell Optical Microscopy Experiments and Image

1st Annual Undergraduate Quantitative Biology Summer School

Module 1: Single-Cell Optical Microscopy Experiments and Image “Fluorescent Labeling Techniques used in single-cell Research. Part 1.1” Lecturer: Linda Forero-Quintero e-mail: [email protected] Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Molecular signals 1. The Central Dogma of Gene activation Molecular Biology 2. Labeling Proteins for Single- Molecule Imaging a. Methods for labeling proteins b. Fluorophores Replication Transcription Translation

Outline-Part 1.2:

3. Labeling Techniques Employed in Fixed cells a. Immunolabeling b. Single-Molecule Fluorescence In situ Hybridization (smFISH) 4. Questions & How can we visualize these processes? Practice 5. Supplementary Material Image modified from the Kahn Academy site (see supplementary material) Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Fluorescence microscopy allows biological processes to be studied as 1. The Central they occur in space and time, at the cellular and molecular levels. Dogma of Taken from Molecular Biology Fluorescence Microcopy book, 2. Labeling edition 2017. Proteins for Single- Molecule Imaging a. Methods for labeling proteins b. Fluorophores 3. Labeling Techniques Employed in Fixed Taken from Fluorescence cells Microcopy book, edition 2013. Jablonski diagram, displaying the energy levels and a. Immunolabeling Fluorescence excitation (dashed lines) and emission possible routes of excitation & de-excitation of a b. Single-Molecule (full lines) spectra of three different fluorophores. The fluorophore. Fluorescence In arrows indicate the laser used to excite these dyes. situ Hybridization (smFISH) Common approaches for protein 4. Questions & labeling Critical properties of fluorescent labels Practice 1. Dye molecules linked to the protein of 1. Location of the fluorophore 5. Supplementary 2. Brightness, blinking & photostability Material interest 2. Genetically encoded fluorescent 3. Location of the protein of interest proteins Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1:  Antibodies are proteins and part of the immune system capable of recognizing intruders like virus and bacteria. Each antibody recognizes a specific antigen. 1. The Central Dogma of Molecular Biology 2. Labeling Proteins for Single- Molecule Imaging a. Methods for labeling proteins b. Fluorophores

Outline-Part 1.2: Image modified from Photometrics

3. Labeling  Biotinylation is the covalently process of attaching biotin to a protein, nucleic acid, Techniques or other molecule. It has high affinity for its interaction partners avidin and Employed in Fixed streptavidin, which can be used to tag fluorescent proteins. cells a. Immunolabeling b. Single-Molecule Image modified from AAT Bioquest Fluorescence In situ Hybridization (smFISH) 4. Questions & Practice 5. Supplementary Material Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1:  Epitope Tags are useful when antibodies are not available. Those can be encoded into a plasmid vector attached to the protein of interest. 1. The Central Dogma of Examples: Molecular Biology 2. Labeling Proteins for Single-  Flag-Tag (AA-Sequence: DYKDDDDK) Molecule Imaging  HA-Tag (AA-Sequence: YPYDVPDYA) a. Methods for  V5-Tag (AA-sequence: GKPIPNPLLGLDST) labeling proteins  b. Fluorophores Myc-Tag (AA-Sequence: EQKLISEEDL)

Outline-Part 1.2: Taken from Photometrics

3. Labeling  Small molecules probes are recruited by a peptide or protein recognition Techniques sequence that is fused to the target protein. Employed in Fixed cells a. Immunolabeling b. Single-Molecule Fluorescence In situ Hybridization (smFISH) 4. Questions & Taken from Fernández-Suárez & Tang, 2008 Practice Examples: 5. Supplementary  Direct Labeling: PolyAsp, HaloTag, SNAPTag, CLIPTag Material  Enzyme Mediated Labeling: SorTag, Qtag, AB, & LAP Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1:  Bioortogonal Labeling occurs between two functional groups (e.g., tetrazines & strained alkenes/alkynes), and requires genetic code expansion. 1. The Central Dogma of Molecular Biology 2. Labeling Proteins for Single- Molecule Imaging a. Methods for labeling proteins b. Fluorophores Taken from Photometrics Taken from Outline-Part 1.2:  Organic dyes are generally planar molecules of Fluorescence Microcopy about 1-2 nm. These dyes belong to different book, edition 3. Labeling 2017. Techniques families based on their core scaffolds. Employed in Fixed cells Examples of commercial organic dyes: a. Immunolabeling  Alexa Fluor posses the largest spectral variety available. b. Single-Molecule Alexa molecules are negatively charged and hydrophilic Fluorescence In situ Hybridization fluorescent. (smFISH)  Cyanines (Cy2, Cy3, Cy5, & Cy7), can be linked to 4. Questions & nucleic acids or proteins through their interactive groups. Practice 5. Supplementary Material Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Taken from Fluorescence Microcopy book, edition 2017.  Quantum dots are inorganic semiconductor 1. The Central Dogma of nanocrystals, typically composed of a Molecular Biology cadmium selenide core and a zinc sulphide 2. Labeling Proteins for Single- shell. For biological applications, these are Molecule Imaging coated with a layer that improves solubility, a. Methods for and then conjugated to targeting labeling proteins b. Fluorophores biomolecules, such as antibodies or streptavidin. Outline-Part 1.2:

3. Labeling Techniques  Minor groove binding dyes bind tightly to DNA in the minor groove region. There Employed in Fixed are about 50 molecules that bind DNA, but they also bind RNA. The only ones that cells a. Immunolabeling have low affinity for RNA are DAPI and Hoechst. Thus, these are commonly used as a b. Single-Molecule nuclear stain. Fluorescence In situ Hybridization (smFISH) 4. Questions & Practice 5. Supplementary Material Taken from Fluorescence Microcopy book, edition 2013. Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1:  Fluorescent proteins are very small and specific, and genetically encoded into the st 1. The Central protein of interest. Green Fluorescent Protein (GFP), was the 1 fluorescent protein Dogma of to be reported. Molecular Biology 2. Labeling What made 1960: Green protein was Proteins for Single- them glow? purified from Jellyfish by Molecule Imaging a. Methods for Shimomura in Japan labeling proteins b. Fluorophores Osamu Shimomura Jellyfish Aequorea Victoria Outline-Part 1.2: 1992: Douglas Prasher Roger Tsien 1994-2016: Roger Tsien mainly 3. Labeling reported the cloning and Techniques nucleotide sequence for wt- contributed to much of our Employed in Fixed GFP in gene understanding of GFP works and for cells developing new techniques and a. Immunolabeling b. Single-Molecule mutants of GFP Fluorescence In situ Hybridization (smFISH) 1994: The coding sequence of fluorescent 4. Questions & GFP is expressed in heterologous cells of Practice 5. Supplementary E. Coli and C. elegans by the lab of Martin Material Martin Chalfie Chalfie Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1:  Fluorescent proteins (GFP) Spectra of GFP variants 1. The Central Dogma of Molecular Biology 2. Labeling Proteins for Single- Molecule Imaging a. Methods for labeling proteins b. Fluorophores

Outline-Part 1.2:

3. Labeling Taken from Olympus confocal web site. Techniques Employed in Fixed Movie of the GFP structure created by cells Erik A. Rodriguez with UCSF chimera in a. Immunolabeling memory of Roger Tsien b. Single-Molecule Most common applications: Fluorescence In situ Hybridization • Reporter assay (GFP as a reporter (smFISH) gene) 4. Questions & • Fluorescence microcopy (Protein Practice folding, protein transport, RNA Discovery, expression 5. Supplementary and development of Material dynamics, among others) GFP Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1:  Bioluminescence is the production and emission of light by a Firefly female 1. The Central living organism on its own. It uses energy from adenosine Dogma of triphosphate (ATP), but does not require light. Molecular Biology 2. Labeling The principal chemical reaction in bioluminescence involves a light- Taken from Wikipedia Proteins for Single- Molecule Imaging emitting molecule and an enzyme, called luciferin and luciferase, a. Methods for respectively. labeling proteins b. Fluorophores Examples of Luciferase based sensors Outline-Part 1.2:

Taken from Ji X. et al 2020. 3. Labeling Techniques Several applications, the most common ones: Employed in Fixed cells • Fluc and Rluc bioluminescence is their use a. Immunolabeling as reporter genes for the study of gene b. Single-Molecule expression in prokaryotic and eukaryotic Fluorescence In cells and systems. situ Hybridization Taken from Syed and Anderson 2021. (smFISH) • Sensors of pH, metal ions, ROS species, 4. Questions & enzymes, drug molecules, among others. Practice • Protein-Protein interaction. 5. Supplementary Taken from Syed and Anderson, 2021. Material • In vivo imaging. 1st Annual Undergraduate Quantitative Biology Summer School

Module 1: Single-Cell Optical Microscopy Experiments and Image “Fluorescent Labeling Techniques used in single-cell Research. Part 1.2” Lecturer: Linda Forero-Quintero e-mail: [email protected] Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: a. Immunolabeling is a biochemical method that allows the detection and localization of an antigen in a cell, tissue or organ, the antigen is usually a protein, and the detection is 1. The Central Dogma of performed using antibodies. Immunolabeling Molecular Biology 2. Labeling Fixation Permeabilization Blocking Antibody Incubation Proteins for Single- Cultured cells (Detergents: Triton x100) (Serum) Molecule Imaging Direct Indirect a. Methods for Sample Chemical Precipitants Detection Detection labeling proteins Preparation Crosslinkers (Metanol b. Fluorophores (PFA) or acetone) Labeled 1ry AB unlabeled 1ry AB Outline-Part 1.2: Can we detect more than one antigen at 3. Labeling a time using this approach? Labeled 2ry AB Techniques Employed in Fixed cells Nuclear counterstaining a. Immunolabeling (DAPI, Hoechst) b. Single-Molecule Fluorescence In situ Hybridization Mounting (smFISH) 4. Questions & Practice Fluorescence 5. Supplementary Microscopy Material Taken from Immunocytochemistry Handbook by Novus Biologicals Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Example of Example of Parallel application of targeting methods immunolabeling & fluorophores 1. The Central Dogma of Molecular Biology 2. Labeling Proteins for Single-

Molecule Imaging Phase a. Methods for Contrast labeling proteins b. Fluorophores

Outline-Part 1.2: Cofilin- 3. Labeling 488 Alexa

Techniques 2006. Employed in Fixed cells DAPI a. Immunolabeling (405) b. Single-Molecule Fluorescence In situ Hybridization (smFISH) 4. Questions & Practice Review Science al. et Giepmans 5. Supplementary Merge Material 20 µm ae from Taken Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: b. Single-Molecule Fluorescence In situ Hybridization “smFISH” (Femino, Singer,

1. The Central 1998) allows the quantification of endogenous transcription response: Dogma of One-layer probes Molecular Biology  Number of individual mRNA per cell, 48 (20bp) probes/mRNA 2. Labeling  3D Location of individual mRNA, Proteins for Single- Tetramethylrhodamine (TMR) Molecule Imaging  DNA transcription site activity, a. Methods for labeling proteins b. Fluorophores

Outline-Part 1.2:

3. Labeling Techniques Employed in Fixed cells a. Immunolabeling b. Single-Molecule Fluorescence In situ Hybridization (smFISH) 4. Questions & Practice 5. Supplementary Material Neuert, Munsky, et al, Science 2013 Munsky, et al, PNAS, 2018 Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: b. Single-Molecule Fluorescence In situ Hybridization “smFISH” (Femino, Singer,

Outline-Part 1.2:

Outline-Part 1.1: b. Single-Molecule Fluorescence In situ Hybridization “smFISH” (Femino, Singer,

3. Labeling Techniques Employed in Fixed smFISH yields highly cells reproducible & a. Immunolabeling b. Single-Molecule quantitative Fluorescence In measurements of situ Hybridization (noisy) single-cell (smFISH) 4. Questions & responses Practice 5. Supplementary Material Neuert, Munsky, et al, Science 2013 Munsky, et al, PNAS, 2018 Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Types of smFISH based on probe design

1. The Central  Traditional smFISH directly targets RNA Dogma of within a cell by using multiple Molecular Biology oligonucleotides (10-50 per target). 2. Labeling Proteins for Single-  Two-layer probes smFISH (like smiFISH), Molecule Imaging indirectly labels the target RNA by a. Methods for labeling proteins fluorescently label a secondary structure b. Fluorophores carried in the primary probe (24 oligos per

Outline-Part 1.2: target are ideal).

3. Labeling  Multiplexing smFISH is Techniques generally used to scale up Employed in Fixed cells the number of RNA a. Immunolabeling targets, and it requires a b. Single-Molecule Fluorescence In parallel on-chip probe situ Hybridization synthesis as well encoding (smFISH) schemes to allow the 4. Questions & Practice identification of bound 5. Supplementary RNAs. Material Taken from Pichon et al. Molecular Cell Review 2018. Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Examples of smFISH, it has been applied to many different RNA in many different organisms. 1. The Central Dogma of Molecular Biology 2 µm 2. Labeling 10 mm Proteins for Single- Molecule Imaging a. Methods for labeling proteins b. Fluorophores 2 µm

Outline-Part 1.2: STl1 mRNA in Ysr35 sRNA in Yersinia c-Fos mRNA (green) and Saccharomyces Pseudotuberculosis (339nt) p-p38 kinase (red) in U2OS 3. Labeling cerevisiae (budding -D. Shepherd cells merFISH detection of 160 Techniques yeast) (LANL / CU Denver) -A. Senecal (CNRS) Employed in Fixed different mRNA species in -G. Neuert (VU) an IMR90 (human fetal cells lung) cell a. Immunolabeling -Chen et al, 2015 b. Single-Molecule Fluorescence In situ Hybridization (smFISH) 4. Questions & Practice 5. Supplementary Material Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: Exercise 1: 1. The Central Dogma of Imagine you need to mark simultaneously three different events in your cells. How do you Molecular Biology choose the right fluorophores, so their emission spectra do not bleed into one another? Use 2. Labeling Proteins for Single- the fluorescence spectra viewer below to determine which combination of fluorophores Molecule Imaging would work to that aim. a. Methods for https://www.thermofisher.com/order/fluorescence-spectraviewer#!/ labeling proteins b. Fluorophores Question 1: Outline-Part 1.2:

3. Labeling Maria is doing a rotation in Prof. Wilson lab, an expert in smFISH. She desires to study Techniques simultaneously the expression of several genes involved in a metabolic pathway. However, Employed in Fixed she is unsure about which type of smFISH to use. Based on what was discussed in this cells a. Immunolabeling module which type smFISH will you advise Maria to try? b. Fluorescence In situ Hybridization (FISH) 4. Questions & Practice 5. Supplementary Material Fluorescent Labeling Techniques used in single-cell Research

Outline-Part 1.1: To learn more about the basis and techniques discussed in this lecture visit the following 1. The Central sites: Dogma of  https://www.khanacademy.org/science/in-in-class-12-biology-india/xc09ed98f7a9e671b:in- Molecular Biology in-the-molecular-basis-of-inheritance 2. Labeling Proteins for Single-  https://www.labxchange.org/library/pathway/lx-pathway:ad7fbf7e-9fee-4989-b8c6- Molecule Imaging e5737d21cc91 a. Methods for  https://www.ibiology.org/online-biology-courses/microscopy-series/fluorescence- labeling proteins b. Fluorophores microscopy/

Outline-Part 1.2: Sources:

3. Labeling 1. Gerd U. Nienhaus & Karin Nienhaus. Fluorescence Labeling. Fluorescence Microscopy, from Principles to Biological Applications. Techniques Editorial Wiley, edition 2013 & 2017, chapter 4. Employed in Fixed 2. Dobrucki and Kubitscheck. Fluorescence Microscopy. From Principles to Biological Applications. Editorial Wiley, edition 2013 & 2017, cells chapter 3. a. Immunolabeling 3. Labeling Proteins for Single-Molecule Imaging by Photometrics. b. Fluorescence In 4. Fernández-Suárez & Tang, Nat. Rev. Mol. Cell Biol., 2008. 5. Ji X. et al. Bioluminescence imaging in mice with synthetic luciferin analogues. Methods Enzymol. 2020, Chapter 8. situ Hybridization 6. Syed and Anderson. Chem. Soc. Rev., 2021, 50, 5668. (FISH) 7. Immunocytochemistry Handbook by Novus Biologicals. 4. Questions & 8. Giepmans et al. Science Review 2006. Practice 9. Neuert & Munsky Science 2013. 5. Supplementary 10. Munsky PNAS 2018. Material 11. Pichon et al. Molecular Cell Review 2018. 12. Chen et al, 2015.