1.

MIKROSCOPE

2. Haemositometer:Manual, software

Dr. Gatot CiptadiLab.Genetika-Pemuliaan ternak

Lab Sentral Ilmu Hayati (LSIH)-UB

Introduction to the Microscope:CarePartsFocusing

1m = 103 mm (millimetres)1m = 106 µm (micrometres)

1m = 109 nm (nanometres)

UNITS OF MEASUREMENT

Eyepiece

Body Tube

Revolving Nosepiece

ArmObjective Lens

StageStage Clips

Coarse Focus

Fine Focus

Base

Diaphragm

Light

• Always carry with 2 hands• Only use lens paper for

cleaning• Do not force knobs• Always store covered• Keep objects clear of desk

and cords

•Light shines through specimen and into a single objective lens and then through the eyepiece (ocular).

•Provides two-dimensional view.

•Specimen must be thin and light must be able to pass through.

•Useful up to about 1,000 X magnification.

•Light shines through specimen and into a single objective lens and then through the eyepiece (ocular).

•Provides two-dimensional view.

•Specimen must be thin and light must be able to pass through.

•Useful up to about 1,000 X magnification.

Compound Light MicroscopeCompound Light Microscope

• Place the Slide on the Microscope

• Use Stage Clips

• Click Nosepiece to the lowest (shortest) setting

• Look into the Eyepiece

• Use the Coarse Focus

• Follow steps to focus using low power

• Click the nosepiece to the longest objective

• Do NOT use the Coarse Focusing Knob

• Use the Fine Focus Knob to bring the slide

Macam-macam mikroskopMacam-macam mikroskop(berdasarkan pencahayaannya)(berdasarkan pencahayaannya)

1.1. Mikroskop cahayaMikroskop cahaya

2.2. Mikroskop stereoMikroskop stereo

3.3. Mikroskop elektronMikroskop elektron

Dissecting Microscope

Dissecting MicroscopeDissecting Microscope

•Light is reflected off a specimen into two different objectives and eyepieces (or oculars).

• Provides a three-dimensional view of object.

•Usually shows surface features of object.

•Useful up to about 60 - 80 X

magnification.

•Light is reflected off a specimen into two different objectives and eyepieces (or oculars).

• Provides a three-dimensional view of object.

•Usually shows surface features of object.

•Useful up to about 60 - 80 X

magnification.

CLSM

Cultured Cell

Live Cell Imaging

Let’s give it a try ...1 – Turn on the microscope and then rotate the nosepiece to click the red-banded objective into place.

2 – Place a slide on the stage and secure it using the stage clips. Use the coarse adjustment knob (large knob) to get it the image into view and then use the fine adjustment knob (small knob) to make it clearer.

4 – When you are done, turn off the microscope and put up the slides you used.

3 – Once you have the image in view, rotate the nosepiece to view it under different powers. Draw what you see on your worksheet!

Be careful with the largest objective! Sometimes there is not enough room and you will not be able to use it!

Carrying a Microscope

1. Rotate the low power objective into place and make sure the stage is all the way down.

2. Place slide on stage making sure object to be viewed is centered over the hole in the stage. Use the stage clips

to hold the slide in place.

3. Turn light on.

4. Focus first with the coarse adjustment knob. Once in focus on low power, turn the nosepiece until the next

higher lens is in place.

5. Use FINE adjustment knob ONLY and focus the object.

Steps to Use:

How to make a wet-mount slide …

1 – Get a clean slide and coverslip.

2 – Place ONE drop of water in the middle of the slide. Don’t use too much or the water will run off the edge and make a mess!

3 – Place the edge of the cover slip on one side of the water drop.

You do not need to use the stage clips when viewing wet-mount slides!

5 – Place the slide on the stage and view it first with the red-banded objective. Once you see the image, you can rotate the nosepiece to

view the slide with the different objectives.

4 - Slowly lower the cover slip on top of the drop.

Cover Slip

Lower slowly

INVERTED MICROSCOPE• Inverted microscope is

widely used for direct observation of cells in cultivation flasks.

• Observe various cell cultures using this microscope. Compare their morphology and density.

Mikroskope elektronMikroskope elektron• Mikroskop elektron mempunyai Mikroskop elektron mempunyai 33 tipe, tipe,

•

– mikroskop elektron scanning mikroskop elektron scanning (SEM) (SEM) untuk studi detil untuk studi detil arsitektur permukaan sel (atau arsitektur permukaan sel (atau struktur renik lainnya), dan obyek struktur renik lainnya), dan obyek diamati secara tiga dimensi. diamati secara tiga dimensi.

– mikroskop elektron transmisi mikroskop elektron transmisi (TEM)(TEM) mengamati struktur detil mengamati struktur detil internal sel. internal sel.

– Scanning Tunneling Microscope ( (STM) ) untuk melihat permukaan untuk melihat permukaan atomatom

Scanning Electron Microscope (SEM)Scanning Electron Microscope (SEM)

•Uses a beam of electrons instead of light.

•The beam of electrons is passed over the specimen and are scattered. These scattered electrons are detected and processed to form an image on a florescent screen.

•Useful up to about ???

•Uses a beam of electrons instead of light.

•The beam of electrons is passed over the specimen and are scattered. These scattered electrons are detected and processed to form an image on a florescent screen.

•Useful up to about ???

Scanning Tunneling Electron Microscope (TEM)

Scanning Tunneling Electron Microscope (TEM)

•Uses a beam of electrons instead of light.•Uses probe and electrons to determine differences in voltage as probe passes over specimen.•Can view objects as small as atoms.

•Uses a beam of electrons instead of light.•Uses probe and electrons to determine differences in voltage as probe passes over specimen.•Can view objects as small as atoms.

UB ?

1. Inverted Mikroskop dan mikromanipulator: FMIPA-BIO, Lab sentral, dan Kedokteran

2. Confocal Laser Scanning Microscope (CLSM): Lab sentral UB; 1. 3 Milyard.)

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

fluorescent (TV) screen,photographic film

Human eye (retina), photographic film

Focussing screen

VacuumAir-filledInterior

MagnetsGlassLenses

High voltage (50kV) tungsten lamp

Tungsten or quartz halogen lamp

Radiation source

x500 000x1000 – x1500Maximum

magnification

0.2nmFine detail

app. 200nmMaximum resolving power

Electronsapp. 4nm

Monochrome

Visible light760nm (red) – 390nm

Colours visible

Electromagnetic spectrum used

ELECTRON MICROSCOPE

LIGHT MICROSCOPEFEATURE

© 2007 Paul Billiet ODWS

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

Copper gridGlass slideSupport

Heavy metalsWater soluble dyesStains

Microtome only.Slices 50nm

Parts of cells visible

Hand or microtomeslices 20 000nmWhole cells visible

Sectioning

ResinWaxEmbedding

OsO4 or KMnO4AlcoholFixation

Tissues must be dehydrated

= dead

Temporary mounts living or dead

Preparation of specimens

ELECTRON MICROSCOPE

LIGHT MICROSCOPEFEATURE

© 2007 Paul Billiet ODWS