5. Light Microscopy (LM) Types

TAMU BMEN 311 - Imaging Living Systems [Light Microscopy Types]

Contrast enhancement

• distinguish fine structural details and visualize transparent or low-contrast specimens more clearly.

• improves visibility of transparent features

specimans examined in LM

• biological tissues and cells

• transparent microorganisms (bacteria and protozoa)

• cellular components like organelles

Types of Light Microscopy

• Brightfield*

• Darkfield

• Phase Contrast

• Differential Interference Contrast (DIC)

• Polarized Light

BRIGHTFIELD

• standard lab microscope

• produces image on a bright background

• Cells and tissues have intrinsically low contrast when imaged in brightfield mode

• using histology stains to improve contrast = toxic → KILLS CELLS

Brightfield applications

routine examination of blood smears, histology, and cytology studies

DARKFIELD

• stars become visible at night

• Increases contrast without staining

• producing a bright image on a darker background

• useful for viewing THIN, LIVE specimens

• sensitive to debris

Darkfield applications

• image syphilis

• studying external details

• tiny diffracting objects like bacteria and isolated organelles

PHASE CONTRAST

• uses refraction and interference caused by speciman’s structures create high-contrast, high-resolution images

• no staining, useful for viewing live specimens.

• utilizes phase shifts caused by differences in refractive indices to enhance contrast in transparent, thin specimens.

• subject to “halo” artifacts

phase contrast applications

nuclei, neurons membranes, cell division, tissue engineering applications

Label the image

1. brightfield

2. dark field

3. phase contrast

DIFFERENTIAL INTERFERENCE CONTRAST (DIC)

• interference patterns → enhance contrast = high-contrast images of living organisms with (pseudo) 3D appearance

• useful in distinguishing structures

• enhances contrast in specimens with slight variations in thickness and refractive index

  • USEFUL FOR THINK SPECIMANS

  • NO “halo” artifacts

  • expensive

DIC Applications

cell motility, cellular structure, cell division (pic), tissue engineering applications

POLARIZING LIGHT

polarized light and filter to study birefringent specimens

Birefringence

optical property of a material

• refractive index depends on polarization and propagation direction of light. (aka double refraction)

Polarizing Light Applications

spindle and collagen fibers (pic), gout crystals, (normal and malaria-infected) red blood cells; optically anisotropic specimens (birefringent)

light polarization