Introduction to the oil immersion compound microscope

Purpose of microscope Microscopy is the technology of making very small things visible to the human eye. Used in the diagnosis of many diseases. Used for soil, insects and rocks. Used in pathological laboratories and medical. Industrial microscope : Used for metals and various kinds of measurement . Educational microscope. Used for research.

Purpose of microscope in Microbiology Bacteria is one of the small microorganisms that can’t be seen with the naked eyes, most bacteria range in size between 0.5-2.0 micrometers (μm) so, there is a need to magnify the bacteria several times by using a microscope in order to see it. There are different types of microscopes which are used in microbial life. In this lab, you will become familiar with the use of the light compound microscope (particularly oil immersion microscopy) and will compare the relative size and shape of various microorganisms.

Basic component of microscope Revolving Nosepiece Nosepiece:  The part of the microscope that holds the objective lenses also called a revolving nosepiece or turret. Body:  This term is used mostly with the low power stereo microscopes and it is the basic heart of the microscope without any type of stand (base) or illuminators.  It usually includes the eyepiece and objective lenses but not the focusing block.

Microscope consists of 4 main parts: frame work. Adjustment system. Magnification. Lighting System.

Frame work Its includes three parts: Base Arm mechanical stage

Mechanical Stage Stage- the location of the specimen to be viewed Clips- utilized in holding the specimen in place

Stage Knobs control Top knob: is to move the forward and backward. Bottom knob: is to move the stage right and left.

Adjustment system It consists of the following parts: Optical tube. Coarse adjustment. Fine adjustment.

Focus and Resolution Parts Course-adjustment knob- is the larger of the two knobs. It is used in bringing the object into quick focus. Fine-adjustment knob- is used for improving the clarity of the image, especially when viewing under high power.

Interpupillary adjustment To control the distance between the ocular lenses to adapt the distance between viewers eyes so, the eyepiece lenses will spread apart or get closer together to fit each individual.

Diopter ring adjustment

Getting in Focus

Magnification Microscope has two sets of lenses are : Ocular lens (eyepiece). Objetive lenes.

Microscope lenses: Ocular lens or eyepiece is used for viewing. Revolving nosepiece contains objective lenses that are used to magnify the image in combination with the ocular lens. The objective of microscopy is not just to increase magnification, but to do so while retaining sufficient resolution.

TOTAL MAGNIFICATION Total magnifigation: Powers of the eyepiece (10X) multiplied by objective lenses determine total magnification.

Depth of focus: thickness of the field; as magnification increased , depth of focus is decreased. Field of vision: the surface area of view; as magnification increased , the area of view decreased.

Oil immersion lens

Refraction and Refractive Index Refraction = bending. Refractive index: measurement of the extent that the substance bend light. Field of view > or equal 500X cause increase in diffraction due to the differences in refractive index between glass and lenses (1.51) and air (1) which increase refraction, and small lenses present in high magnification so that oil is used in these lenses as it has refractive index similar to glass, lenses and specimen which equal 1.51 ,as a result the distortion will decrease.

Resolution The objective of microscopy is not just to increase magnification, but to do so while retaining sufficient resolution. Resolution: It is the ability of microscope, at specific magnification to distinguish two separate objects situated close to one another and the ability of the lens to reveal fine details. The smaller the distance between the two specific objects that can be distinguished apart, the greater the resolution power of the microscope. The depth of focus is how clearly your specimen or object appears to a human eye.

Shorter wavelengths of light provide greater resolution. As magnification increased the resolution also increased. To achieve high magnification with good resolution the objective lens must be small. Shorter wavelengths of light provide greater resolution. Tube Length and Coverslip Thickness. The marks on the line below the the magnification and the numerical aperture are the tube length/coverslip thickness. The mechanical tube length (between the objective flange and the eyepiece seating face) is normally 160 (in mm) for older objective lenses or ( infinity for infinity-corrected objectives). The number after the slash is the thickness in millimeters of the cover glass for which the objective was designed and corrected. For most objectives for close working distance, this number is 0.17. This designation means that you should use No. 1½ coverglasses which range between 0.16 and 0.19 mm in thickness. No. 0, 1, and 2 coverglasses are not recommended. Some lenses will have a - sign. This means that the objective is meant to be used with no coverglass. LD (long working distance) objectives may go up to 1.5 mm so that one may look through slides or tissue culture flasks or dishes. Under the best conditions, with violet light (wavelength = 0.4 um) and a numerical aperture of 1.4, the light microscope can theoretically achieve a limit of resolution of just under 0.2 pm.

A typical cell is 10-20 um Light microscope sees 0.2 um 1 mm=10-3 m 1 um=10-6 m 1 nm=10-9 m 1 A=Angstrom =10-10 m

Numerical Aperture Numerical Aperture (N.A.):  This is a number that expresses the ability of a lens to resolve fine detail in an object being observed.  It is derived by a complex mathematical formula and is related to the angular aperture of the lens and the index of refraction of the medium found between the lens and the specimen. Numerical Aperture (N.A.):  This is a number that expresses the ability of a lens to resolve fine detail in an object being observed.  It is derived by a complex mathematical formula and is related to the angular aperture of the lens and the index of refraction of the medium found between the lens and the specimen.  To get the best possible image, you should have a condenser system that matches or exceeds the N.A. of the highest power objective lens on your microscope.  (note, N.A. is only important with high power microscopes). 

Lighting System: Power supply Transformer Variable resistance Lighting system consists of modern microscopes of the following parts : Power supply Transformer Variable resistance light source sub stage condenser Aperture diaphragm

Lighting System: The Condenser (fucous light through specimen ) Lamp- typically a light source underneath the stage Diaphragm- controls the amount of light allowed to pass through the specimen

How to use Microscope Plug in microscope and turn on illuminator. Rotate nosepiece to lock 4X objective in place Place smear on stage and center it under the 4X objective. Using the course adjustment knob, move the objective lens to its lowest point. Look through the ocular and focus upward with the coarse adjustment until an image comes into view. Rotate nosepiece to obtain the next objective lens 10x and repeat step 3. Rotate nosepiece to obtain the next objective lens 40x . Look through the ocular and focus upward with the fine adjustment until an image comes into view. When using oil immersion lens put a drop of oil on the slide and Rotate nosepiece and repeat step 6.

Factors that influence the quality and size of image: Quality of Microscope and lenses. Size of sample. Type of sample. Amount of light on the sample. Quality of sample.

Microscope Maintenance

Microscope Storage Proper storage of the microscope will prevent or reduce problems! Optics and mechanisms of the microscope must be protected from: Dust and dirt, Fungus. Store the microscope: Under a protective cover, In a low humidity environment. Always carry a microscope with one hand holding the arm and one hand under the base.

Microscope Cleaning Process Cleaning the Eyepiece, Cleaning the Objectives, Cleaning the Microscope Stage, Cleaning the Microscope Body, Cleaning the Condenser.

Cleaning Solutions and Solvents Soap solution for cleaning of body and stage. Ether-Alcohol, Alcohol, or Lens Cleaner Solution for cleaning of lenses. Refer to manufacturer’s guide for appropriate organic solvent.

Cleaning Materials Lint-free cotton gauze pads Lint-free cotton swabs Lens paper Commercial lens tissue for optics Caution: Do not use paper towels or other rough paper products Alternatives include: Fine quality tissue paper Muslin cloth Silk

Microscope Cleaning Process Cleaning the Eyepiece, Cleaning the Objectives, Cleaning the Microscope Stage, Cleaning the Microscope Body, Cleaning the Condenser.

Step 1: Cleaning the Eyepieces Blow to remove dust before wiping lens. Clean the eyepieces with a cotton swab moistened with lens cleaning solution. Clean in a circular motion inside out.

Step 2: Cleaning the Eyepiece Wipe the eyepieces dry with lens paper. Repeat cleaning and drying if required.

Step 3:Cleaning the Objectives Objectives are cleaned while attached to microscope Moisten the lens paper with the cleaning solution Wipe gently the objective in circular motion from inside out Wipe with dry tissue or lens cleaning paper . Be sure to clean the oil immersion objective after use. Objectives should never be removed from the nosepiece.

Step 4: Cleaning the Microscope Stage Wipe the microscope stage using the cleaning solution on a soft cloth. Thoroughly dry the stage. Repeat above steps, if required.

Step 6: Cleaning the Condenser Unplug the microscope from power source. Clean the condenser lens and auxiliary lens using lint-free cotton swabs moistened with lens cleaning solution. Wipe with dry swabs.

Step 5: Cleaning the Microscope Body Unplug the microscope from power source. Moisten the cotton pad with a mild cleaning agent . Wipe the microscope body to remove dust, dirt, and oil. Repeat steps 1–3, if required.

Video on how to use Microscope