4. Light Microscopy Basics

TAMU BMEN 311 - Imaging Living Systems [Light Microscopy]

Light microscopy

• optical microscope

• used to visualize structures and objects too small to be seen by the naked eye

LM input mode

visible light

LM Resolution and Magnification Limits

resolution to ~200nm

magnification to x2000

parts of LM microscope

a light source, condenser, objective lens, and eyepiece (ocular lens)

sample properties

refract, reflect, or absorb based on its properties

objective lens

collects and refracts the light to create a magnified image

ocular lens

eyepiece

Image Formation

• image you see when looking in your microscope is NOT the same as what you would see with your eye

magnified, flipped, and blurred

Why is the image blurred?

point spread function and diffraction

The Point Spread Function (PSF)

• response of an imaging system to a point source or point object

• light passes through an aperture (hole)

• diffraction causes it to spread out

  • causes light from closely spaced objects to overlap

  • lowering spatial resolution

• forming a perfect point, it forms an airy disk (convolution, blurring)

diffraction limit

• min distance between two points to be distinguishable

• image will blur into one, appearing as a single point if closer than diffraction limit

resolution

• depends on the wavelength of light used

• numerical aperture of the microscope’s objective lens

Abbe Resolution Criterion

0.5𝜆/NA

• FOR OBJECTIVE LENS

Rayleigh Criterion (Refined)

0.61𝜆/NA

• FOR OBJECTIVE LENS

numerical aperture equation

NA = n sin 𝛼

• refractive index: n_air = 1.0 and n_oil = 1.4

• visible light wavelength (𝜆): 400nm to 700nm

FOR OBJECTIVE LENS

why add oil

improves resolution by improving numerical aperture by increasing the refractive index

how is resolution affected by Small Objective NA

poor resolution

how is resolution affected by Large Objective NA

excellent resolution

resolution if the condenser aperture

Total Magnification

= (Magnification of Objective) * (Magnification of Ocular Lens)

magnification of Ocular Lens

1X-30X (usually 10X)

magnification of Objective Lens

2X-200X (low 10X, high 40X, oil immersion 100X)

what contributes to image control

condenser and iris diaphram

Condenser

collects and focuses light from illuminator onto specimen, under stage

Iris Diaphragm

controls amount of light that reaches specimen, under stage

• acts as a control for resolution and contrast IN LM

Opening the diaphragm too much

glare and loss of contrast

Closing the diaphragm too much

increased diffraction and loss of resolution

Intermediate position 60-90% opening

optimal resolution and contrast

Aberrations

“distortion of an image” or “lens errors”

• interaction of light with glass lenses

what are the two main types of aberrations

1) Geometrical or Spherical Aberrations

2) Chromatic Aberrations

corrective optics

aberations can be fixed using a series of optical lenses to counteract

Spherical Aberrations

blurring or distortion of image

• light rays from the periphery of lens focusing at different points (before or after) those from the center

Chromatic Aberrations:

blurring or color fringing of images

• component wavelengths (colors) focusing at different points

light path in LM

ocular lens → objective lens → speciman → condenser lans → diaphram