BiMi 2

Bioimaging and microscopy  

Light microscopy  

• Sample preparation  

• Collection, fixation  

• Staining of samples for specific proteins, structures/ organelles or overview staining  

• Light microscopy techniques 

• Lightfield, darkfield  

• Phase contrast 

• DIC 
  fluorescence microscopy  

• Dissection microscopes  

Different types of samples for microscopy  

• Sliced/sectioned samples of tissues or organs 

• Cells in 2D or 3D cultures 

• Smear samples such as blood samples 

• Squashed/spread/smear samples for examining chromosomes (karyotype) in the nucleus  

• Samples consisting of entire small organisms (whole mounts), organs or embryos 

• Live cells or tissue slices  

• Crystals materials etc.  

• Tissue samples are usually 4-10 µm thick – so that light can go through them 

• Specimen are often collected between a microscope slide and a cover glass for light microscopy 

• Cover glass thickness usually 0.17mm 

Preparation of cell samples  

• Attaching cells to coverslip  

• Adherent cells  

• Some cells attach well and grow on glass 

• Otherwise, you can use coated extracellular matrix (eg collagen, fibronectin, matrigel) coverslips or culture dishes 

• Using plastic or glass coverslips  

• Suspension cells  

• Cytospinning 

• Using coated coverslips or culture dishes  

Preparation of “histological” samples usually from tissues  

• Selection and collection of samples  

1. Fixation  

• Cells and tissue needs to be fixed to maintain the natural structure, to inhibit post-mortem degradation and to intensify/enable staining  

• Bacteria that multiply at rapid speed infects the tissue, its own enzymes start to catalyze the breakdown of proteins into amino acids, -> amino acids diffuse out of the cell and prohibits proteins from clotting  

• Should be done as soon as possible (seconds, minutes) after removing a tissue from an organism to inhibit degradation (autolysis)  

• Generally a chemical process in liquid  

General rules for selection and isolation of study object for histology  

• The piece of tissue should not be too big to allow penetration of the fixative  

• Depends on tissue type, fixation, later use etc. few mm often optimal  

• The piece of tissue is not to be allowed to dry and should hence be stored in buffer. The tissue should be treated “physiologically” until fixed 

• The isolated piece of tissue should be placed in fixative or be frozen as soon as possible 

• Avoid tearing and squishing the tissue with scissors and scalpels 

Important properties of fixatives  

1. Rapid penetration ability 

2. Kill quickly and prevent post-mortem reactions 

3. Preserve cell and tissue structure 

4. Stabilise cells and tissues 

5. Making the material harder, but not brittle  

6. Increase the refractive index 

7. Make the material possible to stain  

Characteristics of fixatives  

• There is no ideal fixative! 

• Different fixatives are used depending on the type of tissue and the characteristics/ details desired to study  

• For immune staining of specific proteins, different proteins require different fixations to optimal result and to maintain its actual location etc  

• A fixative may function by  

• Binding proteins – additive 

• Precipitating proteins – coagulating  

• Additive or non-additive of both types  

Fixation methods 

• Immersion fixation  

• The tissue is removed from a dead or alive (biopsy) organism and immediately placed in fixative (most common)  

• Perfusion fixation  

• The fixative is pumped into the bloodstream via the heart or the aorta, so that it rapidly penetrates into the tissue through capillaries. Needed for good fixation of the architecture of some tissues eg. Lung  

Factors affecting fixation  

• Buffer capacity (pH) 

• Penetration capacity (the diffusion constant of the fixative) 

• Volume (ideally 10:1 fixative: tissue) 

• Temperature 

• Concentration 

• Time  

Fixatives  

• Aldehydes 

• Cross-links proteins (especially lysines) 

• Cannot dissolve lipids  

• Formaldehyde, paraformaldehyde (PFA- must be heated to fix and fresh!, often better for immunostaining) and glutaraldehyde (GA) 

• Formalin is formaldehyde usually with methanol  

• Formaldehyde  

• Is routinely used in pathology laboratories at 10% buffered formaldehyde 

• Does not significantly affect protein structure (antigenisity is not affected mostly but sometimes)  

• Good penetration capacity  

• Slow 

• The fixation solution needs to be buffered  

• Most common in pathology  

• Glutaraldehyde 

• Causes deformation of the alfa-helix structure of proteins  

• Bad penetration capacity, pieces need to be very small 

• Fast fixation  

• Good and used for electron microscopy 

• Alcohols 

• Methanol and ethanol (undiluted, no water) 

• Usually at -20°C, 10 minutes  

• Denaturing 

• Causes fragility, hardness and shrinkage  

• Dissolve lipids  

• Are rarely used for whole tissues but often for frozen sections 

• Used for smear samples and cells as they are fast fixatives giving clear nuclear structures 

• Acetic acid 

• The oldest fixative 

• Excellent penetration capacity  

• Can efficiently fix cell nuclei, but not cytoplasmic proteins 

• Does not harden the tissue 🙁 

• Causes swelling 🙁  

• Acetone  

• Precipitates proteins 

• Usually at -20°C, 10 minutes 

• Used for fixing of cells on cover glasses or frozen sections (do not fix cells on plastic, acetone will melt the plastic) 

• Used for enzymatic stainings of unfixed sections  

• Picrates (not used much anymore) 

• Fixatives containing picric acid such as Bouin’s fixative 

• Picric acid cross-links proteins, forming protein picrates that have high affinity for acidic dyes 

• Picric acid cannot be used as such because it severely shrinks the tissue  

• Picric acid does not harden the tissue  

• Oxidizing compounds 

• Permanganate, dichromate or osmium tetroxide  

• Bad penetration capacity  

• Makes the tissue soft (and difficult to slice) 

• Cross-links proteins and causes extensive denaturation  

• Used to study enzymes such as esterases, phosphatases and dehydrogenases by using substrates for these enzymes that reacts with osmium tetroxide  

• Often used for electron microscopy  

• Mercury-based fixatives  

• Precipitate proteins and form mercury bridges between –SH, carboxyl and amine groups  

• Penetrate tissue reasonably well and give excellent staining of the cell nucleus  

• Can leave metal precipitate in the sample, makes it freeze poorly  

2. Embedding and sectioning/slicing  

• Cutting tissue into thin sections/slices  

• Sectioning makes it possible to illuminate the tissue and see the different tissue components with a light microscope 

• To make thin sections of tissue, it must be hardened by: 

• Embedding the tissue in a material that hardens 

• Paraffin wax 

• Plastic mixtures, eg OCT (optical cutting compound) 

• Wax (paraffin) embedding  

• Most routinely done on formaldehyde or paraformaldehyde fixed tissue pieces 

• Embedd/ position sample in molten warm wax (65°C) to a “block” in small plastic cassettes/ molds 

• Avoid air bubbles, surround completely with paraffin  

• Wax becomes solid at RT, can be stored at RT “forever”  

• Before wax can enter the tissue, several steps are needed  

1. Tissue dehydration – water in the tissue needs to be replaced with ethanol, this happens slowly using an ascending ethanol series 

2. Tissue clearing – as ethanol does not mix itself with paraffin wax, the ethanol in the tissue needs next to be replaced with xylene  

3. Tissue infiltration – the tissue is embedded in a block of wax that is let to stiffen  

4. Tissue sectioning – the embedded sample can then be cut into slices with a rotation microtome or a sliding microtome 

5. Tissue rehydration – before staining the tissue, the wax has to be removed with xylene, and the tissue needs to be rehydrated in a descending ethanol series  

Embedding unfixed tissues in plastic “OCT” instead of paraffin  

• Frozen sections can be made from both fixed and unfixed tissue  

• Tissues are frozen down in blocks surrounded by OCT in a block: (OCT=10% polyvinyl alcohol 4% polyethylene glycol), which is a liquid at RT and solid when frozen, OCT needs to surround the entire piece, no bubbles  

• “frozen OCT” embedded tissue is usually cut into 6-20 micron thick slices, with a sharp knife in a device called cryostat 

• The cryostat consists of a rotation microtome placed inside a chamber that is kept chilled (-20°C) 

• The sections can be fixed directly after sectioning in any fixative 

Vibratome 

• It is also possible to cut non-frozen, and even non-embedded and non-fixed tissue with a device called a vibratome  

• The vibratome takes a vibrating sharp blade slowly through the tissue, allowing for slicing of soft tissue  

• This method is slow and gives relatively thick slices (40-50 microns) 

• The method is used in relatively small scale for histological samples  

• Spectrum of visible light= 400-700 nm  

• Visible light, the agent used as the analytic probe in light microscopy, is a form of energy called electromagnetic radiation. This energy is contained in discrete units or quanta called photons that have the properties of both particles and waves  

=> electromagnetic radiation having characteristics both typical pf particles (photons) and of waves  

• The properties of light are determined by 

• Wavelength (λ) = colour  

• Frequency (Hertz) = oscillations/sec 

• Speed of light is 300 000 km/sec. In air  

• Amplitude = intensity/strength of light  

• Oscillation plane or polarity  

• When light hits an object it will absorb some of the light waves of white light and reflect others  

• We see the waves that are reflected as colored light  

• The color we see is the complementary colour to the one that was absorbed  

• An opaque surface can absorb part of the light in its surface, or reflect light  

• Dyes contain at least one benzene ring, one chromophore and one auxochrome in their structure 

Chromophores  

• An atom or group whose presence is responsible for the colour of a compound  

• Side groups that like benzene contain free electrons  

• These side groups are attached to the benzene ring in different combinations 

• Chromophores alter the energy of the electron cloud of the benzene ring to enable absorption of light waves within the visible spectrum  

• Benzene absorbs light waves at 200 nm, our visible spectra is 400-700 nm 

Dye modifiers  

• Methy or ethyl groups alter the colour of the dye by altering the electron cloud of the benzene ring without providing free electrons itself  

Auxochromes  

• Ionize the dye, affecting the absorption capacity of the dye  

• Function as colour enhancers  

• Example: colourless naphthalene becomes strongly yellow when coupled to both chromophores and auxochromes  

Interaction between the dye and the tissue  

• How does the dye bind the tissue? 

• Ionic bonds (positive ions and negative ions attract)  

• Proteins contain positively charged amino groups and negatively charged carboxyl, hydroxyl, phosphate and sulphate groups that attract opposite charged groups in the dye  

• Covalent bonds (shared electrons with dye and tissue, strong!) 

• Dipole-dipole interactions (weak) 

• Van der Waals forces 

• Hydrogen bonds  

Dyes bind based on ions and charged molecules 

• Many dyes attract to ionic radical in the tissue: 

• Basophilic dyes (cationic (+) react to negatively charged ions eg. Nuclei, mucus and cartilage some proteins have amino acids that interact with basic catanionic dye  

• Acidophilic dyes are acidic, anionic dyes e.g. Binds erythrocytes and some leukocytes with free NH_2+ 

• Generally: 

• Acidic dye stains cytoplasm  

• Basic dyes stain chromatin  

Lysochromes = lipid dyes    

• Cannot ionize 

• The staining process is based on the lysochromes being soluble in fat and hence, binds to triglycerides, fatty acids and lipoproteins in fatty tissues  

Mordants (betmedel) 

• Some dyes have low affinity for the tissue and must be used together with mordants  

• Mordant = metal salts that form polyvalent metal ions in solution  

• The mordant and the dye forms a chelate complex together  

• The positive metal ion and, hence the dye can then covalently bind negatively charged carboxyl, hydroxyl, phosphate and sulphate groups in the tissue  

Factors affecting the staining process  

• pH 

• The charge of a protein is dependent on the pH 

• At isoelectric pH(pl), the charge of a protein is zero  

• When the pH decreases below pl, the amount of ionised amine groups increases, and the amount of ionised hydroxyl groups decreases, and the protein becomes alkaline 

• When the pH increases the amount of ionised amine groups is reduced and the amount of ionized carboxyl and hydroxyl groups increased  

• An acidic dye gives stronger staining upon addition of an acid  

• An alkaline dye gives stronger staining upon addition of base  

• pH adjustments are usually small and the pH in the staining solutions is usually 4-8 

Salts  

• Salts in solutions forms positively and negatively charged ions that can bind proteins  

• If both the dye and the tissue is negatively charged, they repell each other  

• If a salt is added, its positively charged ions can bind the negatively charged ions in the tissue. Now the tissue can attract the dye that can bind through for example dipol-dipol interactions 

Phenol  

• Phenol increases the intensity of the staining by reducing the surface tension, which brings the dye and the proteins closer to each other  

• Is also used as a common pH indicator in cell culture  

The solvent 

• Water  

• Most common 

• Very polar 

• Gives rise to maximum ionisation of the dye molecule  

• Ethanol 

• Second most common, often used diluted in waterbn  

• Polar, but not as polar as water  

• Enhances dipol-dipol interactions  

Differentiation of tissue components= removal of excess dye= destaining = washing  

• Different components of a tissue have different amounts of charged groups -> some components are stained faster than others  

• Differentiation can be done with for example the solvent, with pH-adjustments or with mordants  

• The tissue is evenly stained if the staining process is allowed to continue until the tissue is saturated 

• To distinguish between tissue structures, differentiation is required = removal of excess stain 

Regressive vs progressive staining  

• Regressive staining (washing) 

• The tissue is over stained slightly, after which it is destained to achieve the right tone and tissue differentiation 

• More common, faster  

• Progressive staining  

• The tissue is stained until it has reached the right tone of colour and tissue differentiation  

• For this method, very dilute solutions, which may operate for a long time, are used  

• Different tissue components are stained with different intensity leading to differentiation of tissue structures  

• Most dyes are coloured, ionising, aromatic organic compounds + binding enchancing factor  

• Dyes are classified according to whether they are acidic, alkaline, amphoteric or neutral  

Staining methods  

• Haematoxylin is a natural dye extracted from timber  

• Has been used for a long time  

• Haematoxylin is oxidized with sodium iodide or mercuric oxide to form the active dye hematein  

• By itself it binds quite poorly and is most often used together with mordants  

• Alum haematoxylin  

• Aluminum is the most common mordant for haematoxylin  

• Purple in acidic solution 

• Blue in alkaline solution  

Haematoxylin as a dye  

• Haematoxylin together with mordants (e.g. alum haematoxylin) stain cell nuclei very efficiently 

• Can be used for both progressive and regressive staining, depending on how concentrated the solution is 

• In regressive staining the differentiation is performed in a weak acid solution. The acid breaks the bond between the tissue and the mordant.  

• When the staining is complete, the nuclei are colored purple. The nuclei turn blue if the pH is raised a few seconds in an alkaline solution or a few minutes in tap water.  

• The final result is blue cell nuclei and an almost colorless background 

Eosin y  

• Stains the background orange/yellow/pink to easily distinguish it from the nuclei. If some acid is added, the color becomes redder. Stains mainly cytoplasm, red blood cells, muscle collagen. 

• Differentiates between different non-nuclear tissue components in order to distinguish between different tissue types.  

• Is actually classified as a fluorophore according to structure, but rarely used as such.  

Fluorescence  

• Occurs when a molecule emits light at a specific wavelength when exited by light of a shorter wavelength 

• Fluorophores – absorb light, then emits it at a longer wavelength  (some energy is lost as heat) 

Phalloidin – an actin filament marker  

• Phalloidin is a toxin that naturally binds F (filamentous)-actin 

• Phalloidin is a useful tool for investigating the distribution of F-actin in cells 

• Can be conjugated with fluorescent dyes such as FITC, leading to staining of actin filaments  

Immunostaining  

= staining with antibodies specific for specific proteins with fluorescent or non-fluorescent dye detection 

• In fluorescence detection the method is often called immunofluorescence 

Antibodies 

• Are produced by the B cells of the immune response upon exposure to foreign organisms or molecules 

• The antibodies bind the foreign molecules or cells, leading to inactivation, death or phagocytosis  

• The foreign molecules that the antibodies bind are called antigens thus enabling specific binding to a specific protein  

In situ-hybridization  

• Genes are transcribed into mRNA 

• RNA polymerase opens the DNA double-strand and produces an mRNA strand complementary to the gene, processed and transported into the cytoplasm -> protein  

• mRNA can be detected in cells with labelled probes  

The history of the microscope  

• At the turn of the 16^th and 17th centuries 

• Hans and Zacharias Jansen  

• Colors were not refracted properly, pictures deteriorated 

• -> achromatic lens was a major achievement 

• Microscope development took off in the late 1800s, better image quality and resolution  

• First phase contrast microscope in 1938  

Parts of the microscope  

Field diaphragm  

• Adjusts the size of light  

• Desirable to illuminate the whole visual field  

Condenser  

• Simplified, upside down objective  

• Focuses the incoming light on the sample  

• Is corrected for lens faults  

Aperture diaphragm  

• Used to regulate the condenser numerical aperture  

• Best resolution when the condenser and objective NAs correlate  

Objective  

• Consists of several lenses  

• Most important part of the microscope  

• Magnification, resolution and NA 

Chromatic aberration  

• A lens made from glass of a single type  

• Lights of different color are not refracted in the same way  

Spherical aberration  

• The lens refractive index varies with distance from the lens center  

• The middle of the lens does not refract light similarly  

Phase contrast microscopy 

• Cells that seem almost transparent in bright field microscopy can be visualized  

• Phase diaphragm  

Interference contrast microscopy 

• Light is divided into two beams of polarized light  

• Will be very close to each other during their path through the sample 

• Non-stained samples can be studied