Introduction to Microscopy

Question 1

Understand the basic principles of how:

Light
Phase-contrast and differential interference contrast (DIC)
Epifluorescence
Confocal (and other techniques to increase resolution)

microscopes work

Answer 1

1) Light Microscopy:

• uses visible light and magnifying lenses to visualise small structures
• Samples can be live or fixed, and can be stained with various dyes to enhance contrast and identify specific structures

2) Phase-Contrast Microscopy

• This technique enhances contrast in unstained cells by exploiting differences in the refractive indices of cell structures
• It converts phase shifts in light passing through a transparent specimen to brightness changes in the image
• allows for the observation of living cells and their processes without disturbing them with stains or dyes

3) Differential Interference Contrast (DIC) Microscopy:

• produces a pseudo-3D image by using prisms to split light beams, creating differences in light paths that generate shadows and highlights
• It provides more detailed images of internal cell structure than phase-contrast microscopy

4) Epifluorescence Microscopy:

• The sample is illuminated with a specific wavelength of light that excites the fluorophores (fluorescent molecules) in the sample
• The excited fluorophores then emit light at a different wavelength, which is captured to form an image
• Fluorescent markers can be used to label specific structures or molecules within cells, allowing their visualisation and tracking

5) Confocal Microscopy:

• This technique improves the resolution and contrast of the fluorescently labeled specimens by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane
• This leads to the creation of sharp, 2D “optical slices” of the sample, which can be combined to generate a 3D image.

Question 2

Explain why a researcher might use these various types of microscopy (Light, DIC, Epifluorescence and confocal) to:

Recognise images from the different approaches
Understand the basics of epifluorescence
Describe a number of strategies used to facilitate ‘live imaging’

Answer 2

1) Light Microscopy:

• useful for observing the gross morphological features of cells and tissues
• Researchers might use it when they want a broad view of the cellular or tissue structure or to visualise stained specimens
• Light microscopes are also relatively inexpensive and easier to use

2) Differential Interference Contrast (DIC):

• DIC microscopy enhances the contrast in unstained, transparent samples, making it useful for viewing living cells or organisms
• It provides a 3D-like image by generating artificial shadows as if the sample is illuminated from the side
• This allows researchers to observe structures inside living cells without staining, making it an excellent choice for examining cellular processes in real-time

3) Epifluorescence Microscopy:

• This technique uses fluorescent probes to visualise specific structures within cells
• When the probes are excited by light of a certain wavelength, they emit light at a different wavelength, providing high-contrast images of the structures of interest against a dark background
• This method is beneficial when researchers need to study the distribution of specific proteins or other molecules within cells
• It is also frequently used in immunochemistry, where antibodies tagged with fluorescent dyes are used to detect specific antigens

4) Confocal Microscopy:

• Confocal microscopy is commonly used when researchers want to construct detailed 3D images of cells or tissues
• It uses a pinhole to block out-of-focus light
• resulting in ‘optical sectioning’ - the ability to capture sharp, high-resolution images from different depths within thick specimens
• These slices can be compiled to form a detailed 3D reconstruction of the sample

Live Imaging Stratagies:

1. Use of Transgenic Organisms: Researchers often use transgenic organisms that express fluorescent proteins, which can be tracked in real-time using fluorescent microscopy techniques
2. Temperature Control: Many live-imaging microscopes have temperature control capabilities, as maintaining an optimal temperature is crucial for the survival and normal behaviour of living specimens
3. Minimising Phototoxicity and Photobleaching: The use of low light intensities, short exposure times, and specialised dyes can minimise damage to the cells (phototoxicity) and fading of fluorescence (photobleaching)
4. Perfusion Systems: For long-term imaging, systems may be equipped with perfusion apparatuses to provide fresh medium and remove waste, keeping cells healthy throughout the imaging process
5. Time-Lapse Imaging: Taking a series of images over time can allow researching to track changes and movements within living cells