Microscopy Sample Preparation Notes

Sample Preparation for Microscopy

Notes on sample preparation for microscopy, covering fixation, histological and histochemical methods, immunological methods, and in situ hybridization. The quality of images obtained through microscopy heavily relies on optimizing three key aspects: the microscope itself (or other imaging device), the image acquisition/detector settings (such as camera configurations), and the specimen preparation/experimental procedure. Each of these elements plays a crucial role in ensuring that the final image is clear, accurate, and representative of the sample being studied.

Optimizing Imaging

To get great images, optimize these three things:

1. Microscope (or other imaging device).

2. Image acquisition/detector (e.g., camera settings). Proper adjustment of camera settings such as exposure time, gain, and resolution is essential for capturing high-quality images.

3. Specimen preparation/experimental procedure. The way a specimen is prepared significantly affects image quality. Proper fixation, sectioning, and staining are critical.

Overview of Methods

• Part 1: Fixation, histological, and histochemical methods. These techniques are fundamental to preserving tissue structure and enhancing contrast.

• Part 2: Immunological methods and in situ hybridization. These methods allow for the specific labeling and detection of proteins and nucleic acids within the sample.

Visualization and Contrast

Most tissue preparation aims at making the invisible visible. This can range from simple techniques that exploit the physical properties of the instrument, such as dark-field illumination and phase contrast imaging—particularly useful for living cells—to more complex manipulations. The goal is always to enhance the contrast and visibility of the structures of interest.

• Can be very simple/exploit physical properties of the instrument used:

  • e.g., dark-field illumination. Dark-field microscopy enhances contrast in unstained samples by illuminating the sample with light that does not directly enter the objective lens.

  • phase contrast imaging (particularly useful for living cells). Phase contrast microscopy exploits differences in refractive index to produce contrast in transparent specimens, making it ideal for live cell imaging.

• May involve simple tissue preparation but highly complex manipulation of the experimental subject:

  • e.g., expression of fluorescent proteins by transgenic or gene editing techniques. This involves genetically modifying cells to express fluorescent proteins, allowing for the visualization of specific structures or processes.

• May involve highly complex fixation, embedding, sectioning, and staining:

  • Sectioning makes thick things thin enough to transmit light or other energy. It is essential for reducing opacity in thicker specimens, allowing for better light transmission and clarity.

  • Staining or fluorescent tags can label proteins or DNA/RNA/other components. These labels selectively bind to specific cellular structures, enhancing contrast and enabling their visualization.

Sources of Contrast

• “Light”: electromagnetic radiation in the wavelength range $350-750nm$. This range is commonly used in light microscopy to visualize samples.

• Other bands used in specialized bio-imaging techniques (e.g., infrared imaging, X-rays). These are utilized in advanced imaging for specific applications. For example, infrared imaging can penetrate deeper into tissues, while X-rays are used in computed tomography (CT) for 3D imaging.

• Non-EM energy sources:

  • Electrons: Used in electron microscopy to achieve much higher resolution than light microscopy. Electron microscopy uses beams of electrons to create highly detailed images of samples.

  • Ultrasound: Applied in ultrasound imaging for non-invasive visualization of tissues and organs. Ultrasound imaging uses high-frequency sound waves to produce images of internal structures.

  • Magnetic resonance: Utilized in MRI for detailed anatomical and physiological imaging. MRI uses strong magnetic fields and radio waves to generate detailed images of the body's internal structures.

  • Positrons: Used in positron emission tomography (PET) for visualizing metabolic processes. PET imaging uses radioactive tracers to visualize biochemical changes in tissues.

What can create differences in this energy that we can image?

We exploit differences in the absorption (or phase retardation), reflection, or emission of the energy source. These principles are fundamental to generating contrast in microscopy images. For example, differences in light absorption by different cellular structures can be enhanced using specific stains.

Differential Reflection

• e.g., Low power stereo microscopy

• High image power reflected light/epi-illumination (coaxial) commonly used for imaging opaque materials such as minerals. Epi-illumination is valuable for examining surfaces without needing to transmit light through the sample. In epi-illumination, the light source and objective lens are on the same side of the sample.

• Some applications in biology (e.g., SEM). Scanning electron microscopy relies on differential reflection to create detailed surface images. SEM uses a focused beam of electrons to scan the surface of a sample, creating a highly detailed image of its topography.

• Relies on surface structure/absorbance to provide color/luminance difference (contrast). Contrast arises from variations in the surface properties of the sample. Rough surfaces scatter more light, appearing brighter, while smoother surfaces reflect light more uniformly, appearing darker.

• Sample preparation varies – none at all (live imaging) to elaborate (SEM). The preparation can range from minimal for live imaging to extensive for SEM, including fixation and coating. For SEM, samples are typically coated with a thin layer of metal to enhance electron reflection.

Differential Absorption/Transmission

Only works for thin samples!

• Transmitted light microscopy

• Tissue needs to be sectioned or cleared to reduce opacity. Sectioning and clearing are necessary to reduce opacity and allow light to pass through the sample. Clearing involves treating the tissue with chemicals to make it transparent.

• Can be imaged using either phase contrast/DIC

• or after staining tissue to selectively absorb light within structures. Staining enhances contrast by selectively coloring specific structures. Common stains include hematoxylin and eosin (H&E).

Differential Emission

• Fluorescence imaging. Fluorescence imaging involves labeling specific structures within the sample with fluorescent dyes or proteins, and then illuminating the sample with light of a specific wavelength to induce fluorescence.

• Most often using immunolabelling techniques to tag proteins with a fluorochrome (dead/fixed & sectioned or cleared). Immunolabeling uses fluorescent tags to highlight specific proteins, requiring the sample to be fixed, sectioned, or cleared. Antibodies that specifically bind to the target protein are conjugated to a fluorochrome.

• Many techniques for live cells (including functional imaging). Functional imaging allows researchers to observe dynamic processes in living cells. This can include monitoring changes in ion concentrations, protein localization, or enzyme activity in real-time.

Specimens

1. Cells (eukaryotic & prokaryotic)

2. Tissues

3. Organs

4. Parts of organisms

5. Whole organisms

Live or dead (e.g., fixed) specimens?

2-4 typically too thick (opaque) for transmission or fluorescence microscopy without sectioning or tissue clearing

Histology

Histology: preparing tissue for microscopic examination. It involves various techniques to prepare tissue samples for detailed microscopic analysis.

• Thin sections required to resolve cellular detail. Thin sections are crucial for achieving high resolution at the cellular level. Typically, sections are between 5 and 10 micrometers thick.

• New techniques for clearing tissues are beginning to allow high-resolution ‘optical sectioning’ of thicker specimens (e.g., in confocal or light sheet microscopy). Tissue clearing makes the tissue transparent, allowing for deeper imaging with reduced light scattering.

• Sections give access to both surface and internal structures within cells and tissues

• Morphology (shape) already important information. The shape and structure of cells and tissues provide valuable diagnostic information. Pathologists often use morphological features to identify disease states.

• Allow us to exploit other techniques, such as immunolabelling

• Serial sections allow 3D reconstruction (IF you don’t damage or distort the individual slices!) Serial sections are used to create three-dimensional reconstructions of tissues, provided the sections are not damaged during preparation. Specialized software can align and combine the images from serial sections to create a 3D model.

Sections

• Often required to resolve cellular detail

• Quasi 2-dimensional slices through sample

• Fixation of tissue often required. Fixation preserves the tissue structure and prevents degradation. Common fixatives include formaldehyde and glutaraldehyde.

• Tissue needs to be supported to allow sectioning, typically by freezing or embedding in a supportive medium (wax, epoxy, agarose/gelatin). Support is necessary to maintain the integrity of the tissue during sectioning. The choice of embedding medium depends on the desired section thickness and the type of microscopy to be used.

• At high numerical aperture, depth of focus is often too small to see all depths within a single section in sharp focus at the same time. High numerical aperture lenses have a shallow depth of focus, requiring precise focusing to visualize different layers within a section. This is particularly relevant in confocal microscopy.

Fixation: Why?

• Endogenous enzymes degrade proteins: cell structure degenerates rapidly in death…. Enzymes break down proteins post-mortem, leading to rapid tissue degradation. This process is called autolysis.

• Many tissues contain commensal pathogens (e.g., normal ‘flora’). These pathogens can accelerate tissue decay if not inactivated.

• ‘fixation’ is the application of a technique to denature proteins and other cellular processes, whilst preserving cells and tissues (as far as possible!). Fixation aims to halt cellular processes and preserve tissue structure by denaturing proteins. Fixatives create chemical bonds between proteins, stabilizing their structure and preventing degradation.

Aims of Tissue Fixation Parallel the Preservation of Food

• ‘Shelf life’ of cooked /frozen food is extended compared with freshly killed

• Cooking/freezing kills many pathogens, stops normal cellular processes

• Some physical and chemical resemblance to original living sample; but not identical!

• Fixation adds new components & changes or loses others. Fixation alters the chemical composition of tissues, introducing new substances and removing others. For example, formaldehyde can add methyl groups to proteins.

Fixation: An Impossible Compromise

• To some extent fixation is an impossible compromise:

• Fixation destroys normal cellular processes. While preserving structure, fixation inevitably disrupts cellular functions. This can affect the activity of enzymes and the localization of certain molecules.

• Yet many histological techniques target proteins that exist in life….. Many methods rely on identifying proteins as they exist in their natural state. Immunohistochemistry, for example, uses antibodies to detect specific proteins in tissues.

• Different methods aim to retain key protein sub-units (e.g., for identification by immunolabelling) whilst otherwise preserving cellular structure in a ’life-like’ state….. The goal is to maintain essential protein structures for techniques like immunolabeling while keeping the tissue as close to its living state as possible. This requires careful selection of fixatives and fixation conditions.

Fixation Artefacts

Fixation can introduce artefacts into preserved structure:

• Shrinkage/expansion: Tissues may shrink or expand due to the fixation process. This can distort the overall morphology of the sample.

• New protein complexes that may not have existed in the living tissue. Fixation can create artificial protein complexes. These complexes may be recognized by antibodies, leading to false positive results in immunohistochemistry.

• Reaction products: Unintended chemical reactions during fixation can alter the tissue. These reactions can modify proteins and other molecules, affecting their properties.

Always be critical: is what I see a real (i.e. ‘life state’) structure or an artefact?

Fixation Artefacts Examples

Fixation artefacts in preserved structure:

• Heat damage

• Crushing (dissection)

• Autolysis (too slow). If fixation is too slow, autolysis can occur, leading to tissue degradation. Autolysis is the self-digestion of cells and tissues by their own enzymes.

• Poor fixative penetration (too slow). Inadequate penetration of the fixative can result in uneven preservation. This can lead to differential staining and distortion of tissue structures.

Verifying Structure

Fixation can introduce artefacts into preserved structure:

• In some (rare) cases, we may be able to verify structure in living versus fixed samples, e.g. by genetic expression of fluorescent proteins. This involves expressing fluorescent proteins in live cells and then comparing their localization before and after fixation.

• Generally we need to rely on experience and well-tried and trusted methods – many histological processing stages are there for a reason!

Chemical Fixation

Fixatives act by:

• denaturation of proteins. Proteins are denatured to prevent enzymatic degradation. Denaturation involves disrupting the protein's three-dimensional structure.

• cross-linking of molecules (usually proteins, peptides and amino acids). Cross-linking provides structural stability by forming bonds between molecules. This helps to preserve the tissue's architecture.

• Stable meshwork aids subsequent slicing

• fixation primarily affects proteins

• carbohydrates and lipids are difficult to fix…. These are harder to preserve through standard fixation methods. Special techniques are often required to visualize carbohydrates and lipids.

• mono- and disaccharides are not fixable.

• polysaccharides (glycogen, starch) can be ”trapped” within fixed protein meshwork. Polysaccharides can be retained by trapping them within a meshwork of fixed proteins. This allows them to be visualized using specific staining methods.

• lipids can be oxidized by e.g. $OsO_4$ and dichromates (important for electron microscopy where lipid membranes can be resolved). Osmium tetroxide and dichromates are used to oxidize lipids, making them visible in electron microscopy. Osmium tetroxide is particularly useful for visualizing lipid bilayers in cell membranes.

Common Fixatives

Common fixatives:

• Buffered aldehydes most common (e.g. “PFA” = formaldehyde in solution). Buffered aldehydes like formaldehyde are widely used for their effectiveness in preserving tissue structure. Formaldehyde cross-links proteins, creating a stable tissue matrix.

• ‘combination’ fixatives for animal tissue may mix several different components:

  • Examples:

    • Alcoholic Bouin’s solution: picric acid, acetic acid, formalin, ethanol. Bouin's solution is a complex fixative that provides excellent preservation of tissue morphology.

    • AFA: alcohol-formalin-acetic acid. AFA is another combination fixative commonly used for preserving tissues.

• Aldehydes reduce antigenic activity of many proteins, thus reducing efficacy of immunological binding techniques. This can complicate techniques like immunolabeling. The reduction in antigenicity can make it more difficult for antibodies to bind to their target proteins.

• Alternatives: Zinc salt fixatives or non chemical fixation (e.g. microwave)…. Or no fixation at all (‘frozen’ sections). Alternatives include zinc salt fixatives, microwave fixation, or using unfixed frozen sections. Zinc salt fixatives can preserve antigenicity better than aldehydes, while microwave fixation rapidly cross-links proteins. Frozen sections are used when rapid diagnosis is needed or when antigens are particularly sensitive to fixation.

Embedding

• embedding medium depends on desired thickness – thinner sections require a more supportive medium. Thinner sections need a more supportive medium to maintain their integrity. Paraffin wax, epoxy resin, and cryo-embedding media are commonly used.

• dehydration sometimes required (ethanol or acetone series) to replace water with solvents for non-aqueous embedding media (paraffin wax, epoxy monomers). Dehydration is often necessary to replace water with solvents that are compatible with non-aqueous embedding media. This prevents distortion of the tissue during embedding.

• even thicker sections possible using vibrating blade microtome (“Vibratome”) using embedding media that match tissue firmness without dehydration (e.g. gelatin/albumin or agarose gels). Vibrating blade microtomes allow for thicker sections using media that don't require dehydration. This is useful for preserving delicate structures or for immunohistochemistry.

Paraffin Wax Sectioning

• Knife (metal) imperfection & softness of paraffin wax limits the minimum thickness and resolution. The softness of paraffin wax and imperfections in the knife limit the achievable resolution. Sections are typically 5-10 micrometers thick.

• Allows sections in the $3-20<br>ot{m}$ range - suitable for transmitted light microscopy

• Embedding v. quick

• sections can be collected as a serial ribbon, allowing subsequent 3-d reconstruction

• Suitable for large to medium tissue blocks

• Standard for routine histology (clinical pathology, research)

Cryosectioning

• Frozen sections in the $5-25<br>ot{m}$ range, suitable for transmitted light microscopy

• Fixed or unfixed tissue can be sectioned

• Large samples (e.g. whole rodent brain)

• Embedding ‘cryopreserving’ gel is highly compatible with sensitive antigens

• Can even use unfixed samples for enzyme localization etc.

• Faster than paraffin, but section quality worse

• common for routine immunolabelling

• Many applications in pathology

Epoxy Resin Embedding & Ultramicrotome

• thin and ultrathin sections:

  • $0.5$ to $5<br>ot{m}$ for high resolution transmitted light microscopy,

  • $30-70nm$ for transmission electron microscopy (TEM)

• requires high quality fixation/strong cross linking (glutaraldehyde, $OsO_4$)

• slow embedding (days) to infiltrate tissue with resin monomer

• small block size (typically <2 mm)

• V. hard knives – glass, sapphire or diamond

Epoxy resin embedding/Ultramicrotomy

• Gives the highest quality images for Light microscopy ($1<br>ot{m}$ sections)

• Only technique compatible with very thin sections for TEM ($50-70nm$ sections)

Transparency and Contrast

• Thin samples are good for high resolution imaging

• Thicker samples – inhomogeneous refractive index and imperfect match for immersion oil introduces scattering and spherical aberration

However: Inherent transparency means that thin structures lack contrast…

Standard Histological Staining Protocol

1. Remove embedding medium from sections

2. Rehydrate/rinse sections

3. Staining protocol – sequential steps

4. Dehydrate

5. Mounting medium + cover glass

Improves contrast between different tissue components/organelles by exploiting simple binding affinity between stain and intracellular chemicals

Basic or acidic dyes that react with different chemical groups

Dye Staining

Standard combination: Two dyes:

• nuclear dye (basic) +

• cytoplasmic dye (acid)-

• (e.g. haematoxylin-eosin, ”H & E”) - Hematoxylin stains acidic structures (like DNA) blue, while eosin stains basic structures (like proteins) pink.

Many special combinations: ‘Polychrome’ dyes:

• nuclear dye (basic) +

• cytoplasmic dye (acid)-

• (e.g. Azan, KOLW, Masson’s trichrome…etc. etc. etc.) - These dyes stain different structures in various colors, making it easier to distinguish between them.

Special Techniques

Special techniques: can be applied to tissue ’block’ before sectioning, or afterwards

• Golgi impregnation:

  • Beautiful high quality single neuron stains - The Golgi stain stains entire neurons, allowing for detailed visualization of their morphology.

  • Very random! - The Golgi stain only stains a small proportion of neurons, making it difficult to predict which cells will be stained.

• Reduced silver (Bodian stain)

  • Reveals axons and neurites - The Bodian stain is used to visualize nerve fibers, including axons and dendrites.

Histochemical Reactions

Special techniques: exploit residual biological activity of preserved cellular components

• identify specific chemical groups that determine molecule type and/or activity

• make use of specific chemical activity to identify molecules in tissue or cells

• Enzyme histochemistry

  • identifies and locates specific enzymes, e.g. acetylholinesterase, cytochrome C oxidase - This technique uses specific substrates to detect the activity of enzymes in tissues.

• Paget’s ”neurosecretion” stain

  • Chromogenic detection - This stain is used to visualize neurosecretory granules in endocrine cells.

Chromogenic Detection

• enzymatic label catalyzes the conversion of a colorless chromogenic substrate to produce a colored precipitate

• enzyme may be endogenous or applied via histochemical (or immunological) tag

• choice of chromogen directed by the enzyme employed in the experiment

• e.g. AEC (red), DAB (brown) used to detect horseradish peroxidase (or … false positive endogenous peroxidases!)

• “histochemistry”

  • AEC (3-Amino-9-ethylcarbazole),

  • DAB (diaminobenzidine)

Immunological Methods & In Situ Hybridization

• Immunohistochemistry (IHC) = immunocytochemistry (ICC) - These techniques use antibodies to detect specific proteins in tissues (IHC) or cells (ICC).

• (IHC – tissues; ICC – cells)

• Protein is ‘tagged’ by an antibody that is then visualized by either chromogenic reaction or fluorescence

• RNA/DNA detection

• In situ hybridization - This technique uses labeled probes to detect specific RNA or DNA sequences in tissues.

  • Similar to IHC but targeting RNA/DNA rather than protein

  • More commonly uses chromogenic detection

  • Fluorescent tags also used: "FISH"

Thick Sections & Whole Mount

• useful for e.g. neuronal visualization / tracing etc.

• tissue thickness limits penetration of reagents (requires detergent to permeabilise)

• Special tissue clearing agents can make thick tissue transparent for confocal or light sheet imaging

Immunofluorescence and Immunohistochemistry

Immunofluorescence and immunohistochemistry: detection of proteins, peptides and amino acids

• use of specific antibodies (immunoglobulins, usually IgG) raised in lab mammals (e.g. rabbit, mouse etc.)

• a specific molecular configuration recognized by the antibody (amino acid sequence, 3D-structure etc) is called an epitope

• direct or indirect visualization of bound antibodies

• Marker molecule is e.g. a fluorochrome (=fluorophore) or probe for enzymatic detection (chromogen)

Indirect Visualization by Fluorescence = Immunofluorescence

Indirect visualization by Fluorescence = Immunofluorescence

• Primary antibody (IgG) is recognized by secondary antibody raised in different species

• Secondary AB is labelled with a fluorochrome

Visualization of Immunolabelled (Tagged) Proteins by Fluorescence

• expression within tissue: i.e. which cells?

• expression within cells: i.e. where? which organelles?

• different markers allow study of co-localization of more than 1 protein within organelles (optically tricky because of chromatic aberration !)

Visualization of Immunolabelled Proteins

Visualization of immunolabelled (tagged) proteins can also be by

• Enzyme reaction = Immunohistochemistry

• Gold nanoparticles – permit electron microscopy

• Radionuclides – usually used for quantification

Visualization by Enzyme Reaction

Visualization by enzyme reaction, example: Peroxidase-antiperoxidase (PAP) complex

• Enzyme + substrate + chromogen = colored precipitate

• Can be used to amplify weak signal (e.g. low levels of protein expression)

• Less prone to photobleaching than fluorescence, but…

• Less useful for identifying / localizing multiple different target proteins

Production of Antibodies

• Polyclonal antibodies

  • Immunization induces antibody production of many clones of lymphocytes – many epitopes recognized by antiserum

• Monoclonal antibodies

  • Antibody production by selected single clone – only one epitope

• Hybridomas: inject specific antigen into e.g. mouse, collect antibody-producing cell from the mouse's spleen, and fuse it with a tumor cell (myeloma). Hybridoma cells multiply indefinitely - can be used to produce a specific antibody indefinitely.

• Immunization against antigen.

• Several animal species used in antibody (IgG, IgM, IgY) production.

• Rabbit, goat, sheep, guinea-pig, donkey, chicken…

• Mouse, rat, rabbit…

WHY different species?

Multiple Immunolabelling

Multiple immunolabelling:

1. Rabbit anti-YFP (primary)

   Goat anti-rabbit IgG, fluorophore conjugate for detection (secondary)

2. Mouse anti-α-synuclein (primary)

   Goat anti-mouse IgG, fluorophore conjugate for detection (secondary)

Permits simultaneous detection of 2 (or more!) antigens. e.g.:

• Different filters can be used to excite fluorescence (typically) sequentially

• Using appropriate filters in viewing path allows visualisation of each fluorophore

Combining Immunolabelling with Fluorescent Stains

Multiple indirect immunolabelling can be combined with other fluorescent stains: e.g. antibodies against 2 antigens + DNA (DAPI stain):

• DAPI - A fluorescent dye that binds to DNA, allowing for visualization of nuclei.

• FITC- A green fluorescent dye commonly used to label antibodies.

• Texas Red - A red fluorescent dye commonly used to label antibodies.

Options for Immunolabelling

Options depending on antigen:

• heat sensitive?

• dissolves or lost in polar or non-polar solvents?

• All can reduce antibody binding

• Frozen sections or paraffin? $60°C$ for paraffin

• Acetone or ethanol

• Xylene (or substitute) – non polar solvent for wax

Optimization of Immunolabeling

1. Establish optimal concentration of

   • Primary antibodies

   • Secondary antibodies

Many protocols are developed through hard won experience – follow the recipe carefully!

2. Specificity controls

   • Pre-immune serum (polyclonal antibodies) or isotype control (monoclonal antibodies)

   • Pre-absorption with antigen

   • Western blot

In Situ Hybridization

Detecting nucleotide sequences:

• mRNA in cells or in tissue

• DNA on chromosomes

Sample preparation for in situ hybridization:

• Fixation & preparation of tissue sections is basically identical to that for immunolabelling.

• But: RNAase-free environment & solutions!

Detecting mRNA in Tissue Workflow

1. Obtain nucleotide sequence for mRNA of interest (clone & sequence, or bioinformatics)

2. Produce complementary cDNA probe labeled with a hapten* (e.g. Digoxigenin)

3. Hybridize to tissue

4. Detect using variety of methods (similar to indirect immunolabelling)

   *A molecule with high antigenicity (allows selective immunolabelling with very low background ‘noise’)

Visualization of mRNA with In Situ Hybridization

• Autoradiography (radio label)

• Enzyme reaction (needs chromogen)

• Digoxygenin-immunohistochemistry (needs chromogen)

• Fluorescence (‘FISH’) fluorophore-conjugated cDNA, or indirect fluorophore attachment

Advantages / Disadvantages

Advantages / disadvantages of chromogenic versus fluorescence detection: