Microscopio Invertido
Como su nombre lo dice un microscopio invertido está al revés comparado con un microscopio convencional. La fuente de luz y el condensador están sobre la plataforma apuntando hacia abajo.Los objetivos y la torrecilla están debajo de la plataforma apuntando hacia arriba. Las únicas cosas que están en disposición corriente son el tubo binocular o trinocular, así mismo la muestra es colocada sobre la plataforma o platina mecánica.
Aunque un microscopio electrónico tiene gran significado en la magnificación y resolución de la muestra, éste requiere que el especímen sea completamente preparado, conduciendo a que cualquier tipo de vida en la muestra no sobreviva. Por otra parte, el microscopio de luz concede una observación temporal de muestras vivas, sin embargo, éstas requieren una hidratación constante que las somete a estres permanente, imposibilitando de tal manera su supervivencia. A diferencia de estos microscopios, el microscopio invertido permite observar organismos o tejidos en cultivo sin una preparación previa, favoreciendo el monitoreo del estado de crecimiento, comportamiento y demás parámetros involucrados en el desarrollo del cultivo.Con un microscopio invertido se pueden observar cultivos enteros o grandes muestras bajo estados más naturales y con disminuidas condiciones de estres. La observación se realiza a través de las bases de diferentes recipientes con espesores y características ópticas variables, tales como placas de Terasaki, frascos Corning, frascos Roux, cajas de Petri plásticas y de vidrio. El material plástico es ópticamente superior que el de vidrio debido que son más delgados y uniformes.
Una desventaja del microscopio invertido es su limitada magnificación, debido a que los objetivos más potentes son de 40X y de 60X, aunque éste último eventualmente se encuentra en el mercado y es de muy alto costo. Si embargo estos objetivos tienen la ventaja de sobrepasar el límite del grosor del recipiente y observar la imagen con fineza.
http://www.microscopy-uk.org.uk/mag/artjul98/invert.htmlComponentes.
En el microscopio invertido se identifican los siguientes componentes:
Portalámpara: Es un casquillo dentro del cual se encuentra la lámpara que es la fuente de luz halógena.Soporte de filtros: Es un contenedor dentro del cual se insertan filtros específicos para el tipo de iluminación requerida en la observación. El filtro de cobalto es de color azul, pasa la luz de amarillo a blanco, viéndose la imagen en campo claro (es un tipo de iluminación que permite observar la muestra oscura contra un fondo claro). El filtro verde es indispensable para el contraste de fases (es un tipo de iluminación que diferencia la imagen en grado de tono, brillo o color, observándose las células traslúcidas) y eventualmente puede emplearse para contraste en relieve o de interferencia (que produce alto contraste, imágenes tridimensionales y reales de objetos transparentes, a diferencia del contraste de fases suple una claridad definida, imagen detallada de células gruesas o células con amplios índices de refracción).
Portanillos de luz: Es una corredera de anillos, que se maneja de acuerdo con el tipo de iluminación requerida (contraste de fases o contraste en relieve). No se emplean anillos para campo claro.
Apertura del diafragma: Es un dispositivo con el cual se gradúa el nivel de luz emitido por la lámpara. Puede ser mantenido bajo para enfocar fácilmente sobre muestras no coloreadas o abierto para contraste de fases.
Condensador: Es un sistema de lentes que recoge los rayos de luz y los converge dentro de un foco. Está localizado directamente por encima de la plataforma del microscopio y está acoplado con la apertura del diafragma para controlar el incremento de la resolución, realzar el contraste, reducir el resplandor y garantizar óptimos resultados con todas las combinaciones ocular–objetivos.Soporte: Es el que sostiene el condensador y la lámpara manteniendo la estabilidad del microscopio.
Plataforma mecánica: Platina metálica donde se colocan las muestras a observar, presenta un orificio central que comunica la luz proveniente del condensador con los objetivos.
Carro mecánico: Soporte hueco en el cual se acopla el recipiente del cultivo, posee una regleta que permite el movimiento del carro sobre la plataforma mecánica, de derecha a izquierda o de arriba hacia abajo. De acuerdo con el tipo de recipiente se emplea un soporte específico. En el caso de los frascos no se emplea este soporte. Sin embargo éste es un accesorio que puede ser reemplazado, colocando cualquier recipiente directamente sobre la plataforma mecánica y haciendo el monitoreo del cultivo manualmente, con movimientos diagonales o en zig zag.
Objetivos: Es un sistema de lentes complejos localizados debajo de la plataforma mecánica. Los objetivos corrientes de alto poder, tienen muy corta distancia de trabajo y por lo tanto deben acercarse a la muestra para su enfoque, en el microscopio invertido los objetivos son adaptados para trabajar a largas distancias debido a que el grosor de los recipientes impide el contacto estrecho entre el objetivo y la muestra. Además los objetivos son corregidos para enfocar muestras sobre diferentes bases.
El aumento de los lentes se identifica con un código de colores; 4X - rojo, 10X – amarillo, 20X – verde, 32 o 40X – azul. El objetivo de 100X no es disponible para este microscopio. Comercialmente los microscopios invertidos tienen dos presentaciones de objetivos; la primera con aumentos de 4X, 10X, 20X y 32X y la segunda con aumentos de 4X, 10X, 20X y 40X.Revolver de portaobjetivos: Es un dispositivo donde se ensamblan los objetivos.
Otras piezas: Son los botones micro y macrométrico, tubo binocular o trinocular (en caso de poseer un fototubo para adaptar cámara fotográfica), oculares, anillo de ajuste de dioptrías (para la corrección de la diferencia de dioptrías entre ambos ojos), botón para ajustar el bulbo de voltaje (regula la entrada de voltaje al microscopio prolongando su tiempo de duración), receptáculo para el cable de conexión comunicado con el fusible del microscopio.
Algunas ventajas y desventajas del microscopio invertido.
Una gran ventaja del microscopio de luz sobre el microscopio electrónico es la habilidad para observar organismos vivos y tejidos. Varios tipos de microscopios electrónicos requieren que el espécimen altamente preparado (lo cual incluye recubrimientos con oro) y vacío para la observación. En los microscopios de luz se pueden observar temporalmente organismos vivos, lo cual permite determinar algunas funciones vitales, sin embargo las muestras se preparan con soluciones de alcohol o formaldehido, componentes que denaturan las proteínas y los ácidos nucleicos(5). Mientras un microscopio electrónico tiene una alta magnificación y resolución que un microscopio de luz (algunos producen espectaculares imágenes tridimensionales), sólo produce imágenes de muestras muertas(4).
A diferencia de estos microscopios, en el invertido los cultivos o muestras se pueden colocar en diferentes recipientes con espesores y características ópticas variables, se utilizan entonces, placas de Terasaki, frascos Corning, frascos Roux, cajas de Petri plásticas y de vidrio, lo cual permite observar organismos o tejidos en cultivo sin una preparación previa, se pueden además observar grandes cultivos o muestras bajo estados muy naturales, lo cual permite disminuir las condiciones de estrés(4).
La primer desventaja es el costo. Los microscopios invertidos no están en cualquier lugar, como si lo está un microscopio con una configuración estándar por eso es poco competitivo para los fabricantes (4). Otra desventaja es su limitada magnificación, pues el objetivo más potente es de 60X, siendo poco frecuente en el mercado y de muy alto costo. Actualmente se están comenzando a fabricar objetivos de 100X que se utilizan en aceite de inmersión, sin embargo son bastante costosos y escasos.
Inverted Microscopes
by David Goldstein, Seattle, Washington, USA
Inverted microscopes have some advantages and disadvantages compared to a conventional microscope. However, if you can overcome the major disadvantage (the cost of the thing), the advantages are substantial for observing living microorganisms.
Introduction
Recently, I became the proud owner of an Olympus CK2 Inverted Microscope with the result of some damage to my bank balance. However, I find myself using it more and more often in preference to a conventional microscope. The reasons for this are discussed in this article. The Olympus CK2 with and without a trinocular head are illustrated in the photograph to the right. I have the model to the right with the trinocular tube.
I should preface my comments by the limitations on my knowledge. I am hardly an expert on the subject of inverted microscopes. My entire experience with inverted microscopes is limited to the several weeks I have spent with my CK2 (which I understand Olympus is no longer making in preference to some newer and more expensive CK models). I have not tried other inverted microscopes although I have a brochure for Olympus' much more expensive infinity corrected (IX) models.
What is an inverted microscope?
As the name suggests, an inverted microscope is upside down compared to a conventional microscope. The light source and condenser are on the top above the stage pointing down. The objectives and turret are below the stage pointing up. The only things that are "standard" are that (1) a specimen (as dictated by the laws of gravity) is placed on top of the stage and (2) thank heavens, the binocular or trinocular tube is not upside down but in the standard position pointing at a conventional viewing angle. As a result, one is looking up through the bottom of whatever is holding the specimen and is sitting on the stage rather than looking at the specimen from the top, typically through a cover glass, as on a conventional microscope.
The following illustration is taken from the owner's manual and identifies the various components:
What are the Advantages of an Inverted Microscope?
Before getting to the advantage of an inverted over a traditional light microscope, bear with me for a moment while I discuss the advantage of a light microscope over an electron microscope because the inverted microscope carries this advantage one step further. The critical advantage of light microscopy over electron microscopy is the ability to observe living organisms and tissue. The various types of electron microscopes require that the specimen be thoroughly prepared (which may include coating it with gold) and placed in a vacuum chamber for observation. Obviously, whatever life is in the specimen does not survive this process. Light microscopes allow one to observe a live microorganism such as a protozoan (now protist) as it goes about its various life functions. While an electron microscope has significantly greater magnification and resolution than a light microscope (and some types can produce spectacular 3D images), it only produces a snapshot in time of a dead subject.
As anyone who watches microorganisms for minutes and hours at a time can testify, the moving picture that unfolds before the observer of a live organism can reveal much about the organism and its behavior that the momentary snapshot of a dead organism will never show, no matter how detailed. Some organisms change their shape so much from one second to the next that photomicrographs taken seconds apart of a live organism may look like they are of different organisms. It is for this reason among others that light microscopy is well and alive as a research tool despite the advantages of electron microscopy.
A traditional light microscope requires that the specimen be placed on a glass slide, typically under a cover slip (although there are objectives designed for use without a cover slip). This usually means removing a small sample from the culture and placing it in the artificial environment created by the slide and cover slip. The temperature and oxygen content of the sample may change quickly from that of the culture as a result. Further, the organisms will be under increased pressure and in an unnaturally confined space as a result of the cover slip. Also, the sample will quickly dry out unless repeatedly replenished with water. The loss of water by evaporation and the periodic adding of water may change the salinity of the sample frequently. These changes impose severe stress on microorganisms that can affect their behavior and/or kill them in a short time.
Some partial solutions to these problems include making a small chamber on the slide or using a slide with a well or chamber built in and sealing it to prevent evaporation. While this keeps the sample from drying out quickly, the inability of the sample to exchange gasses with the air means that within a day, a few days or a week, the organisms will die. Another partial solution is to use the hanging drop technique in deep well slide which prevents pressure and reduces confinement but again is fairly temporary.
Therefore, observations through a standard microscope are also limited in time. Its images are not the frozen image of an electron microscope but neither can it easily allow study over long duration (it is true that another technique available is to use special reservoir slides with built-in water chambers that allow the specimen to remain active for long periods but this still requires specimen preparation and creates a relatively limited environment for the life on the slide.
Would it not be nice is if you could observe microorganisms in a large container under more natural conditions. This is what the inverted microscope allows and by doing so, it extends the advantage of the light microscope. Because of its configuration, You can place an entire culture or large sample in a relatively large container such as a petri dish and look at the entire contents of the container under more natural (although still admittedly artificial) and less stressed conditions. Such a sample may sustain life over a much longer period. The cover of the container slows evaporation greatly while at the same time allowing some gas exchange. The larger quantity of water is less subject to quick temperature changes although obviously, if stored in a room, the water will acquire the room's temperature. One sample of pond "scum" I have looked at over several weeks still sustains life although the character of that life has changed significantly over time from when I first collected the sample. During this period, as the environment in the petri dish changed, the life that it could support changed but there is still a great deal of varied life.
Since inverted microscopes are often used for looking at living organisms and tissue that may be killed by staining, they often provide for "optical staining" through the use of phase contrast or DIC. My CK2 has phase contrast. Rather than use the more sophisticated phase contrast condenser used on a standard microscope, there is a simple slider that goes through the condenser and holds the necessary phase rings. Only the phase rings for the lower power objectives (e.g. 10x and 20x) are centerable by a fairly crude process (although it works). The phase ring for the 40x objective is not but seems to work fine.
What are the Disadvantages of an Inverted Microscope
The first disadvantage is cost. Inverted microscopes are not anywhere near as common as a microscope with a standard configuration so there is less competition both in the new and used markets. Further, they are more complex and therefore expensive to build. One has to get an image that is pointing down from underneath the stage up to the eyepieces in front of the microscope and pointing up. One does not have to be an optical engineer to see the complexities of this. Further, unlike a standard microscope where focusing is done by simply moving the stage or entire optical tube up and down, at least on my CK2, only the turret assembly moves up and down. All of this adds complexity and cost to the microscope.
On a standard microscope, higher power objectives are optimized for a specific thickness of the cover glass which is quite thin and uniform. With an inverted microscope, you may be looking through the bottoms of different containers with various thicknesses and variable optical characteristics. With higher power objectives, it is advantageous to be able to correct for the differences in thickness and on my 40x objective, there is a correction collar which allows you to correct for container thicknesses over a wide range (correction collars are also available on 20x objectives although mine does not have one and the images seem fine). However, again this adds to the complexity and cost of the objective.
Also, remember that standard high power objectives typically have a very short working distance and must get very close to the subject to focus. This is why they are often protected by a retracting nosepiece to avoid damaging the objective if it accidently comes in contact with the cover slip and slide. Because of the greater thickness of the bottom of the container you are looking through (compared to a cover slip), a standard higher power objective may not be able to get close enough to the subject to focus. Therefore the higher power objectives on an inverted microscope must be corrected for a much longer working distance. These objectives are designated at least by Olympus as "LWD" (long working distance) and "ULWD" (ultra-long working distance) objectives. Again, this adds to the cost. Further, even with all these corrections, they cannot make up for the relative lack of optical clarity and uniformity of looking through a good cover slip and therefore, the quality of the image may not be as good as looking through a conventional microscope with comparable objectives (note that plastic disposable petri dishes are usually more suitable than most glass petri dishes because they are thinner, more uniform and therefore optically superior to the standard glass dish).
However, an inverted microscope is also excellent for viewing pond life under a cover slip. The slide is inverted with the slip facing the objective (the cover slip stays attached to the slide despite the downward pull of gravity because of the surface tension created by the drop of water). This eliminates the compression problems experienced with upright stands. In addition, protozoa and other invertebrates that normally move along the substrate quickly move to the upper surface of the slip, providing a much better optical link with the objective. In fact, this is one reason the inverted microscope is such a hit with cell biologists.
The condenser also must allow for an unusually long working distance to allow larger containers to be placed on the stage and on the CK2 is designated as ULWD. I assume as a result of the necessary correction, it has an "N.A." (numerical aperture) of only .3 and the 20 watt halogen light source which is more than adequate on my standard microscope at high power is just adequate with the 40x objective. This makes photomicrography more difficult. I wish the CK2 had a 30 watt or higher light source (as do Olympus' more expensive models). Surprisingly, I have not noticed much difference in brightness or quality of the image between viewing a petri dish with and without its cover (even with condensation on the inside of the cover). As a result, I tend to leave the cover on when I am viewing.
Another disadvantage is that the maximum magnification available on an inverted microscope is more limited. Typically 40x is the highest powered objective (although Olympus has a 60x objective available on its more expensive model). Oil immersion 100x objectives are often not available (although Olympus has one for its IX series of infinity corrected inverted microscopes). I do not know whether this is because of the difficulty of maintaining an oil immersion drop upside down or the difficulty of correcting such a higher power objective for long working distances.
Finally, a mechanical stage is an extra-cost option on my model and finding petri dishes or other containers that exactly fit the holders that come with it are a little bit of chore. However, one can become adept after some practice at moving the container by hand without the mechanical stage even at high power. This allows following a microorganism more easily through a diagonal or zigzag course through the immensity of a 100 mm diameter petri dish that may have a culture with a depth of several millimeters.
However, whatever disadvantages an inverted microscope has, these are far outweighed by the fundamental advantage of an inverted microscope to accept a container with a large and relatively long-lived diverse culture of live organisms without any preparation.
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