Light microscopy is an indispensable microscopic technique in biological research that employs standard compound microscopes. It allows minimally invasive, three-dimensional observation of the small organisms or objects to analyze the morphological details. It also aids in the study of biomolecules and biological processes. All the microscopic methods using electromagnetic radiation to achieve magnification are included in the light microscopy. Light microscopy is ideally suited for imaging biology because of the well-matched resolution, a diverse range of fluorescent probes, and the non-perturbing nature of light that enable the imaging for longer periods.
The light microscope was invented by Zacharias Jansen (1580–1638) in 1595. He used two lenses for detailed magnification of the object. He employed collapsing tube as microscopes like a telescope in reverse and magnified the items by nine times. Then, Antony van Leeuwenhoek (1632–1723) invented the simple microscope in 1670 with a magnification up to 200x and doubled the resolution as compared to the best compound microscopes of those days. He observed the individual cells, protozoans, bacteria, muscle cells, and sperm for the first time. Englishman Robert Hooke (1635–1703) added a stage to hold the specimen, an illuminator, and fine focus controls to advance the compound microscope further. The magnification was limited to 30x to 50x until the 1800s, and the images exhibited blurry edges and rainbow-like distortions. The issue of the blurry image was resolved by Carl Zeiss (1816–1888) and Ernst Abbe (1840–1905), who added the sub-stage condenser and developed the superior lenses that provided improved resolution and higher magnification.
Light microscopy adds a vital extra dimension to the understanding of cell anatomy and physiology and enables the study of the dynamic cellular events using the live cell fluorescence microscopy.
The light microscope works on the principle of refraction. An image of the object is formed by the objective lens with a magnification of 10x to 100x. This magnified image is then observed through the ocular with a magnification of 10x. The total magnification of a microscope is the sum of objective and eyepiece magnifications. Most broadly, the light microscopy technique can be divided into two categories: fluorescence and brightfield. In the fluorescence microscopy, fluorescent probes are used to observe the cells and tissues of interest. Fluorescent dyes are absorbed by the molecules that absorb one wavelength of light and emit the second, longer wavelength of light. The emitted light then projects the image of the object. In brightfield microscopy, the light source and objective lens are placed on opposite sides of the sample. As the sample absorbs, scatters, or deflects the light, the image is formed. The two most commonly used techniques to visualize the phase shift in light microscopy are phase contrast and differential interference contrast (DIC). The phase contrast causes the cells to appear dark against a light background, and the DIC gives a pseudo–three-dimensional (3D) shaded appearance to the cells. The brightfield microscopy without phase contrast or DIC is sufficient to observe the general outlines of the cells, but phase contrast or DIC is vital to get detailed, high-contrast images (Thorn, 2016).
The light microscope uses visible light to produce the magnified images of the specimens projected onto the imaging device. It comprises two lenses: the objective lens and the ocular that work together to produce the final magnified image. The objective lens collects the diffracted light from the specimen and forms a magnified real image at the real intermediate image plane. The condenser lens focuses the light coming from the illuminator onto the area of interest on the specimen. Other components of the microscope include the tube, the eyepieces, the lamp collector and the lamp socket, filters, retarders, polarizers, and the stage and stand with focus dials (Murphy, 2001).
- Mount the specimen on the slide with the coverslip. Hold the slide with the stage clippers. The slide may require manual positioning to adjust the eyepiece onto it.
- Optimize the lighting to provide high-intensity illumination at high magnifications. Adjust the lighting and brighten the field without hurting the eyes.
- Adjust the condenser and position it with the lens. If the condenser has selectable options, set it to a bright field.
- Focus, locate, and center the specimen.
- Start with the lowest magnification and gradually focus the area of interest on the specimen.
- If the specimen is unstained, then use dark field mode or high contrast.
- After locating the specimen, adjust the contrast and intensity of the illumination
- Raise the magnification in steps. Higher magnification lenses should be physically closer to the specimen.
- Observe and analyze the specimen.
Protocol for Studying Photorespiration (Khoshravesh., Lundsgaard-Nielsen., Sultmanis., & Sage., 2017)
- Cut the leaf with a razor blade.
- Fix the tissues on the dissecting scope stage with a drop of fixative in a fume hood.
- Immerse the tissues in fixative (glutaraldehyde) containing vial.
- Fix the tissue at room temperature for a minimum of 2–3 hours and a maximum of one day.
- Replace the fixative with two 30-min washes of sodium cacodylate buffer.
- Decant the sodium cacodylate buffer from the vial and add 1 mL of fresh buffer. Add OsO4 to the vial and make final concentration to 1% or 2% OsO4. Leave it for 2–3 h at room temperature.
- Decant the OsO4 from the vial and wash the sample with sodium cacodylate buffer for 2-30 minutes.
- For dehydration, decant sodium cacodylate buffer with the help of a transfer pipette and add 10% EtOH (Ethyl alcohol).
- For each successive 10% increase in EtOH, continue decanting the old solution and replace with the new solution until the two changes of 100% EtOH.
- Remove the dehydrating agent and infiltrate the specimen with resin (Spur’s or Araldite resin).
- Preheat oven to 600C and clean the flat silicone embedding molds with acetone. Dry the molds.
- Fill the wells of molds 75% with fresh resin.
- Transfer the infiltrated tissue to each well.
- Fill the tissue-containing wells with resin.
- Place the mold in the oven for 8–16 hours for Spur’s resin, and 48–72 hours for Araldite.
- Turn off the oven and cool the samples.
- Remove the samples from the oven when fully cooled.
Trimming and Sectioning
- Mount the tissue upright in the microtome.
- With one edge of the razor blade, remove small amounts of resin around the tissue.
- Place the trimmed tissue block into the microtome.
- Position the microtome knife.
- Fill the boat with distilled water.
- Set the section thickness to 3 μm and begin the sectioning.
- Place a drop of distilled water on the adhesion glass slide.
- Position an acetone-cleaned glass rod on the side of the floating section and roll the section onto the glass rod.
- Release the section into the water drop by rolling the glass rod.
- Place the glass slide with the tissue sections on the hot plate set to 45–48O
- Flood the slide on the hot plate with toluidine blue with the help of a syringe.
- Remove the slide from the hot plate after a metallic halo forms around the edges of the stain.
- Rinse off the dye with distilled water.
View and observe the sections under the light microscope.
Measurement of Biofilm Thickness (Bakke. & Olsson., 1986)
Biofilm thickness is vital to the understanding of both the theoretical analysis and the practical application of biofilms because of its role in mass transfer. The study was conducted to determine the influence of specimen refractive indices on the thickness of transparent biofilms using the light microscopy. The thickness of the biofilm was measured by measuring the vertical displacement of the sample which moves the focal point across the film thickness. It was found that the mechanical thickness of the biofilm is proportional to the vertical displacement, but not equal to it. It was concluded that the proportionality coefficient depends on the ratio of the refractive index of the measured film to the refractive index of the medium interfacing the film between the film and the objective lens. The biofilm thickness can be measured using the light microscopy if the refractive index is known to the researcher.
Imaging Single Biomolecules in Living Cells (Pinaud. & Dahan., 2011)
Light microscopy allows visualizing single molecules to advance the understanding of biomolecular mechanisms in cells. The individual biomolecules in living cells were targeted specifically and imaged under the light microscope. In this study, complementation-activated light microscopy (CALM) is used in which proteins are fused with dark split-fluorescent proteins (split-FPs), which are then activated into bright FPs by complementation with synthetic peptides. In cells, site-specific labeling of the fluorescent probes is then verified by single microscopy detection with the complemented split-FP fusion proteins. Light microscopy is simple and combines advantages from genetically encoded and synthetic fluorescent probes to enable the researchers to visualize single biomolecules with high-accuracy in living cells, independent of their expression level.
Demonstration of Morphological Changes in Lipodermatosclerosis (LDS) and Venous Ulcers (Tronnier., Schmeller., & Wolff., 1994)
In the study, light microscopy was used to delineate the morphological changes in lipodermatosclerosis (LDS) and venous ulcers. Tissue samples were taken from the patients with trophic skin changes in chronic venous insufficiency. These affected tissues were compared with the normal tissues under light microscopy. It was observed that the ulcer tissue and LDS skin had dilated tortuous vessels in a glomerulus-like arrangement in the dermis. Ultrastructurally, the superficial vessels were surrounded by a cuff with amorphous and basal membrane material. In the deeper parts of the dermis, the vessels of both the ulcer tissue and LDS were surrounded by cellular cuffs with fibroblasts, pericytes, and compact collagen bundles. It was concluded that the severe morphological changes in LDS and ulcer tissue are vital to the pathogenesis of venous ulceration.
The light microscope is a fine instrument which needs proper care and handling. Following are the precautions to be followed while handing the light microscope;
- Clean the microscope with lens paper only by holding the paper flat and parallel to the lens surface.
- Do not remove condensers, objectives, and oculars from the microscope.
- Carefully focus the lens while observing the slide to avoid the slide damage.
- Avoid putting fingers on the lens as it may blur the vision.
Strengths and limitations
- The light microscope is affordable and easy to use imaging tool for tissue sections and microorganisms.
- It is lightweight and small that make it portable for the researcher.
- Using the visible light as the source, the light microscopy provides high-quality images of the specimen observed.
- As the light microscope can maximize the image 1500 times, it may not be sufficient to observe the specimens smaller than 0.275 microns.
- The internal processes in the living cells are difficult to observe without dyes under the light microscope.
- Pinaud., & Dahan., M. (2011). Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins. Proc Natl Acad Sci, 108(24).
- Tronnier., W. Schmeller., & Wolff., H. H. (1994). Morphological Changes in Lipodermatosclerosis and Venous Ulcers: Light Microscopy, Immunohistochemistry and Electron Microscopy. Phlebology, 9, 48-54.
- Murphy, D. B. (2001). Fundamentals of light microscopy and electronic imaging. Canada: John Wiley and Sons.
- Bakke., & Olsson., P. Q. (1986). Biofilm thickness measurements by light microscopy. Journal of Microbiological Methods, 5, 93-98.
- Khoshravesh., V. Lundsgaard-Nielsen., S. Sultmanis., & Sage., T. L. (2017). Light Microscopy, Transmission Electron Microscopy, and Immunohistochemistry Protocols for Studying Photorespiration. Methods Mol Biol, 1653, 243-270.
- Thorn, K. (2016). A quick guide to light microscopy in cell biology. Mol Biol Cell, 27(2), 219-222.