Microscopy Learn how to use a light microscope Identify the parts and its functions Define resolution and magnification Identify different kind of cell and its components

The Microscope

Correct use and care:

transport and lenses

The Microscope

Each type of microscope has a specific function, which makes them suitable for different types of tasks.

Fig. 1: dissecting, fluorescence, phase-contrast and electron microscopes.

The Microscope

Ocular

Binocular tube Arm

Revolving nosepiece Objectives

Condenser

Lamp

Base

Coarse focus adjust.Fine focus adjust.

Mechanical stage adjustment (x,y)

Brightfield

Light is sent up from under the stage Light goes through condenser and is focused

on an object Light goes through object, into objectives and

object is magnified Light goes through oculars to eye and is

magnified again

The Microscope

Magnification: number of times an object is magnified by the lenses

TM = Ocular x Objective

Resolution: the ability of a microscope to show to separate dots as discrete units

The Microscope

Lenses:

“Low power" objectives: are very useful to get overview images of larger sections.

“High power" objectives: are mostly for details and there are more delicate (dry and wet lenses).

As the magnification increases in the lenses, the working distance decreases.

The Microscope

Lenses:

The Immersion lens (100X) is used to increase resolution.

Generally used with oil since its refractive index is very close to that of glass, avoiding lost of light.

The distance between the front lens and the cover glass, also called working distance (WD), can be as small as 0.1 mm.

The Microscope

Estimating size: we need to determine the diameter of field of view for the objectives and estimate the number of times that the object could fit on the area.

Lo = Df or Lp / # of objects

Lo = length of the objectDf = diameter of field of viewLp = length of the pointer (typically ½ of Df)

The Microscope

Estimating size:

Lo = Df or Lp / # of objects

Objective Df (mm)

Scanning 3.0

Low power 1.2

High dry 0.3

Oil immersion 0.1

Table 1: standard set of measurements for Df for common objectives of many microscopes.

Estimating size with microscope

Pointer

Diameter

•Df or Lp/# of objects =Lo

Ex: if Df =0.1mm and there are 2 objects…?

.01mm/2 = 0.05mm/object

ExampleImage 1: plant cells of Elodea sp. in aquarium water

N

CM

Cl

CV

CW

Plant cells Elodea sp. 400X Elodea sp. cell 0.075mm

Cell Theory Cells are the basic units of life All living organisms are composed by cells Prokaryotic vs. Eukaryotic cell Animal vs. Vegetal cell

Biodiversity

Living organisms are organized into

3 domains

The Cell

Cells are the smallest units that exhibits all the characteristics of living organisms

Fig.2: squematic diagram of prokaryotic and eukaryotic cell

Biodiversity

Today, we will see examples of 2 of them

2 domains

The Cell

Prokaryotic cell: Bacteria

Identify rod-shaped and coccus-shaped bacteria