Name : Prakhar Maurya

Entry Number: 2022CSB1102

Group Number: 4
Ge109 Report
Title:Inverted Microscope

Objective: To prepare a detailed report on the working of inverted

microscope and its applications.

Working Principle:
 Light Path Reversal: In an inverted microscope, incident light
emanating from a source above the specimen traverses through
the specimen, undergoing potential absorption, scattering, and
phase shifts, before reflecting off the sample's surface. The
reflected light then retraces its path, traveling back through the
objective lens and the eyepiece assembly.
 Objective Lens: The inverted microscope's objective lens, a
combination of precisely ground glass elements, facilitates the
collection, and magnification of the transmitted or reflected
light waves emanating from the specimen. This process involves
the manipulation of wavefronts, polarization, and diffraction
phenomena to achieve optimal resolution and contrast.
 Condenser: Situated beneath the stage, the condenser
assembly serves to channel and concentrate incident
illumination onto the specimen with precise control over its
intensity, coherence, and angle of incidence. By employing
optical elements such as lenses, apertures, and filters, the
condenser optimizes the spatial and spectral characteristics of
the illumination, thereby enhancing image contrast and clarity.
 Image Formation: Within the inverted microscope's optical
path, the objective lens plays a pivotal role in the formation of
the magnified image. By virtue of its complex optical design and
specialized coatings, the objective lens manipulates the incident
 light waves, converging them onto the focal plane of the
specimen. This convergence results in the formation of a
spatially resolved image, characterized by features
corresponding to the fine structural details and optical
properties of the specimen. Subsequently, the magnified image
is projected upward through the eyepiece assembly, where
additional optical elements further refine its spatial and
spectral properties for observation and analysis.

Brief History/Evolution: The 1950s saw the advent of inverted

microscopes, initially tailored for metallurgical exploration, offering a
novel approach to examine bulky metallic specimens. By positioning
the light source and objective lens above the specimen, metallurgists
gained unprecedented insight into grain boundaries and defects. By
the 1970s, biologists recognized the versatility of inverted
microscopes for studying living cells cultured on vessel bottoms,
revolutionizing cell biology. Advancements in optics, phase contrast,
and DIC microscopy refined imaging capabilities, while the
integration of fluorescence microscopy unveiled intricate cellular
dynamics. In the digital age, CCD cameras and image analysis
software further enhanced capabilities, enabling high-resolution
imaging and quantitative analysis. The history of inverted
microscopes is a testament to continual innovation driven by the
evolving needs of scientists, from metallurgy to cellular biology,
unlocking the mysteries of the microscopic realm.
Components:
 Base and Stand: The sturdy base provides stability, while the
stand holds the optical components.
 Illumination System:
 Light Source: Typically a halogen lamp or LED.
 Condenser: Focuses light onto the specimen.
 Field Diaphragm: Controls the illumination field.
 Objective Lenses:
 Long Working Distance (LWD) objectives are
essential for imaging through thick culture vessels.
 Magnification Ranges: Common magnifications
include 4x, 10x, 20x, 40x, and 100x.
 Stage and Stage Clips: Holds the specimen slide or culture dish.
 Focus Mechanism:
 Coarse Adjustment Knob: For initial focusing.
 Fine Adjustment Knob: For precise focusing.
 Eyepieces: Binocular or trinocular for observation.
 Camera Port (Trinocular Models): Allows attachment of cameras
for documentation.
 Phase Contrast or DIC (Differential Interference Contrast)
Optics: Enhances contrast in transparent specimens.
 Filters: Used for fluorescence microscopy.
 Mechanical Stage: Allows precise movement of the specimen.
 Nosepiece: Holds multiple objective lenses.
 Body Tube: Connects the eyepiece to the objective lenses.
Detailed Working with Schematics:
 Specimen Preparation: Before placing the specimen on the
microscope, it undergoes meticulous preparation. This typically
involves cutting, mounting, polishing, and sometimes etching
the metallic sample to reveal its microstructure. The specimen
is then securely fixed onto a microscope slide or specialized
sample holder.

 Specimen Placement: Once prepared, the specimen is carefully

positioned on the stage of the inverted metallurgical
microscope. The stage may feature mechanical controls to
 precisely manipulate the specimen's position, ensuring optimal
alignment and stability for subsequent imaging.

 Illumination Adjustment: The inverted metallurgical microscope

employs a sophisticated illumination system to illuminate the
specimen from above. This illumination is often provided by a
halogen or LED light source coupled with a condenser assembly.
The condenser allows for precise control over the intensity,
angle, and coherence of the incident light, crucial for achieving
optimal contrast and resolution in the observed image.

 Objective Lens Selection: The microscope is equipped with a

range of objective lenses, each offering different magnification
levels and numerical apertures. The nosepiece of the
microscope allows for seamless selection and rotation of the
desired objective lens based on the specific requirements of the
examination. Higher magnification objectives enable detailed
analysis of fine microstructural features, while lower
magnification objectives provide a broader field of view.

 Focusing Mechanism: To achieve sharp and clear focus on the

specimen, the inverted metallurgical microscope features both
coarse and fine focusing mechanisms. The coarse focus knob
facilitates rapid movement of the objective lens along the
optical axis, allowing for initial approximation of focus.
Subsequently, the fine focus knob enables precise fine-tuning of
focus to resolve fine details within the specimen's
microstructure.
  Observation and Analysis: With the specimen properly
illuminated and focused, the observer peers through the
eyepiece of the microscope to examine the microstructural
features of the metallic sample. Depending on the specific
requirements of the analysis, various contrast-enhancing
techniques may be employed, such as brightfield, darkfield,
polarized light, or differential interference contrast (DIC)
microscopy. These techniques reveal different aspects of the
specimen's microstructure, aiding in the identification of grain
boundaries, phase constituents, defects, and other critical
features.

 Documentation and Recording: Inverted metallurgical

microscopes often feature built-in or attachable imaging
systems, such as digital cameras or CCD cameras, enabling the
capture of high-resolution images and videos of the observed
specimens. These images serve as valuable documentation for
analysis, interpretation, and presentation purposes.

Applications :

In metallurgy, inverted microscopes are essential for analyzing

metallic microstructures, including grain boundaries and defects.
They aid in assessing material quality, composition, and integrity,
crucial for optimizing manufacturing processes and ensuring material
performance in industrial applications. By providing detailed insights
into metal alloys, inverted microscopes facilitate informed decision-
making in materials science and engineering.
Other uses are :
 Cell Culture Research: Studying cell behavior, growth, and
interactions.
 Live Cell Imaging: Visualizing dynamic processes in living cells.
 IVF (In Vitro Fertilization): Assessing embryo quality.
 Tissue Engineering: Evaluating tissue constructs.
 Drug Screening: Testing drug effects on cells.
 Microinjection: Injecting substances into cells.
 Cancer Research: Investigating cancer cells and drug responses.