# Microscopy

• Created by: LBCW0502
• Created on: 10-11-18 12:29
What are the three broad areas microscopy is used?
Clinical environments, research and pharmaceutical manufacturing (process analysis technology)
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What are some of the main uses of microscopy in pharmaceutical analysis?
Estimation of particle size distribution, analysis of particle shape/morphologies, study of distribution of components (API) in formulations, determination of crystallinity of materials, identify some unknown particulates
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What are the basic components of a microscope?
Objective (collect light from sample). Condenser (focus light onto sample). Light source (provide illumination for sample). Eyepiece/camera (visualise image). Same set of lens can act as objective/condenser at the same time (reflection mode)
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What is magnification?
How many times bigger an image is relative to the object. Dependent on focal length of lens and distance between lens and object. Based on objective and eye piece
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How do you calculate the total magnification?
Magnification of objective x magnification of eye piece (actual magnification needs to be verified by standard sample/stage micrometer. Images from microscopy must have scale to show actual dimension)
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What is the quality of an image limited by (despite high magnification)?
Resolution - ability to identify two separate points as two entities. Resolving power if dependent on numerical aperture of objective and condenser (n - refractive index, theta - angle of incidence of outer ray) and wavelength of light used
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What is the equation for resolving power?
r = 0.61 lambda / NA (sin theta - half angle)
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What are the other parameters to be considered?
Working distance (distance between objective and sample that a focus will be achieved) and cover slip thickness used
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What are the common types of microscopy for pharmaceutical analysis?
Polarised light microscopy, thermal microscopy, SEM and micro-spectroscopy (near infrared, infrared, Raman)
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What is a polarised light microscope?
Light microscope equipped with two linear polarisers, one below the condenser, another on top of the eyepiece. Aim to detect a change in polarisation of light upon passing through sample. Used for detection of birefringement crystalline materials
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What is birefringence?
A property of a material where the refractive index (ratio of speed of light in vacuum in the medium) is a function of the polarisation of light
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Describe the process of using polarised light microscopy to analyse crystals (1)
1st polariser generates linear polarised light, 2nd polariser/analyser (90 degrees to 1st polariser) to detect birefringence. Crystalline material will result in a change in polarisation of light
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Describe the process of using polarised light microscopy to analyse crystals (2)
If no crystalline material in sample, no light will pass through the crossed polarisers. A method for detection/enhance contrast of crystalline materials, useful to determine refractive index, thickness and crystal orientations
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What is thermal microscopy?
A combination of thermal study with microscopy. Study of polymorph - different polymorphs can have different m.p. and order of m.p. often is an indicator of order of stability of crystal
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Describe features of a SEM (1)
Non-optical method (use electron beam instead of photon). No eyepiece. High resolution (1-10 nm). High depth of view (3D image). Analysis of topography/size/shape of particles at nanoscale
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Describe features of a SEM (2)
Electrons/particles have wave properties (E=hc/lamba). Energy on the order of keV producing nm wavelength. Electrons focused to small spot and scanned across sample
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When using SEM, what does the magnification and resolution depend on?
Distance between each data point but resolution is limited to wavelength of electron beam (1 nm - can lead to diffraction/blurred image)
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Give examples of application of SEM
SEM images of erythrocytes (see rough/infects RBCs). Haematoxylin and Eosin stain. View samples for malaria infection
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Describe features of microspectroscopy
Spectroscopic measurement at a microscopic level. Spectrophotometer combined with light microscope. For IR, all reflective objective and condenser are used. Variety of applications e.g. API-matrix distribution and drug polymorph analysis
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Describe features of infrared spectroscopic imaging
Infrared spectrometer with focal plane array detector. Each pixel corresponds to unique spatial region on sample. Sample, condenser (IR spectrometer), objective, FPA detector, imaging, (peaks indicate analyte - caffeine/starch/HPMC, amorphous)
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Why are FTIR images limited in resolution (despite high magnification)?
Due to the wavelength of IR (too large)
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## Other cards in this set

### Card 2

#### Front

What are some of the main uses of microscopy in pharmaceutical analysis?

#### Back

Estimation of particle size distribution, analysis of particle shape/morphologies, study of distribution of components (API) in formulations, determination of crystallinity of materials, identify some unknown particulates

### Card 3

#### Front

What are the basic components of a microscope?

### Card 4

#### Front

What is magnification?

### Card 5

#### Front

How do you calculate the total magnification?